Method for detecting purity of modified nucleoside monomer and separating key impurities by adopting reversed-phase high-performance liquid chromatography

Abstract

The invention discloses a method for detecting the purity of a modified nucleoside monomer and separating key impurities by adopting reversed-phase high-performance liquid chromatography, which comprises the following steps: selecting a reversed-phase C18 chromatographic column with octadecylsilane chemically bonded silica or modified octadecylsilane chemically bonded silica as a filler, controlling the detection wavelength to be 260 nm and the operation time to be 35 minutes, and detecting the purity of the modified nucleoside monomer by adopting the reversed-phase high-performance liquid chromatography. Gradient elution is selected as an elution procedure; sequentially injecting a blank solution, a 0.001 mg / mL LOD solution, a 0.002 mg / mL LOQ solution, a standard resolution solution, a reference substance solution, an impurity 1 stock solution, an impurity 2 stock solution, an impurity 3 stock solution and a test solution. According to the method, the purity of the modified nucleoside monomer is detected, key impurities are separated and analyzed, factors such as intermediate precision, specificity, limit of detection, limit of quantitation and reproducibility are investigated, operation is easy and convenient, and the purity and impurities of the modified nucleoside monomer can be better analyzed.

Description

Technical Field

[0001] The present invention relates to a method for analyzing the purity of modified nucleoside monomers, and specifically to a method for detecting the purity of modified nucleoside monomers and separating key impurities by reversed-phase high performance liquid chromatography. Background Art

[0002] In the field of biomedicine, small nucleic acid drugs are considered potential candidate drugs for treating various diseases due to their unique molecular structure and therapeutic mechanism. In recent years, the research and development of nucleic acid drugs has become one of the hottest fields globally. As a representative of nucleic acid drugs, small nucleic acid drugs have shown great potential. Compared with traditional small molecule drugs and antibody drugs, small nucleic acid drugs can intervene from the source, have the characteristics of "treating both the symptoms and the root causes", as well as advantages such as fast target screening, high treatment efficiency, low drug toxicity, strong specificity, and high R & D success rate, making the research and development of small nucleic acid drugs remain highly popular in recent years. However, the research and development process of small nucleic acid drugs is complex and full of challenges. Among them, nucleoside monomers, as the key raw materials for synthesizing small nucleic acid drugs, have a significant impact on the research and development process.

[0003] Nucleoside monomers are the key raw materials in the synthesis of small nucleic acid drugs. They can combine with different bases through specific chemical reactions to form small nucleic acid molecules. The phosphoramide bond in nucleoside monomers has good stability and can resist enzymatic degradation in the body. Therefore, introducing nucleoside monomers into small nucleic acid drugs helps to improve the stability and biological activity of small nucleic acid drugs. With the continuous development of nucleoside monomer synthesis technology, more and more new nucleoside monomers have been developed, providing more possibilities for the research and development of small nucleic acid drugs. These new nucleoside monomers have higher reactivity and better stability, enabling the design and synthesis of more complex small nucleic acid molecules.

[0004] Naturally occurring small nucleic acids generally cannot be used as drugs due to reasons such as their structure being easily degraded by nucleic acid enzymes in the body and poor pharmacokinetic properties. Chemical modification of nucleotides at multiple sites is required. The modification of nucleotide monomers mainly focuses on the sugar ring, phosphate backbone, and base, with the aims of: a) introducing chemical modifications to enhance the stability of oligonucleotides; b) increasing their affinity for targets and promoting cell uptake; c) improving their in vivo bioavailability. For example, introducing 2'-position chemical modification monomers can change the biological properties, chemical stability, and biological activity of nucleic acids, thus providing more possibilities and choices for the function, structure, and pharmacological properties of synthetic nucleic acids. For example, replacing the OH at the 2-position of nucleoside monomers with F, methoxy, oxyethoxymethyl, or tert-butyldimethylsilyloxy, after such nucleoside monomers are used in oligonucleotides, they can improve the affinity of oligonucleotides for complementary RNA and enhance their resistance to nucleases, and they are used in both siRNA and aptamers.

[0005] Nucleoside monomers that meet quality requirements are the development foundation for nucleic acid drugs, mRNA vaccines, primers / probes, plasmid synthesis, etc., and are the key factors for the commercialization of small nucleic acid drugs. Nucleoside monomers often need to be chemically modified after synthesis before subsequent use. Their synthesis involves multiple technical steps. After modification, the molecular structures of nucleoside monomers become complex and diverse, and there are many types of impurities. Since any impurity related to the nucleoside of the nucleoside monomer may spread throughout the synthesis process, there are strict quality control standards in quality control, such as purity, water content, etc. Due to characteristics such as complex processes, high input costs, personalized requirements, and strict production and transportation standards during the synthesis process, it has a relatively high industry barrier.

[0006] Due to the characteristics of modified nucleoside monomers, such as being difficult to vaporize and having a high boiling point, gas chromatography methods cannot be used to detect their purity.

[0007] It should be noted that the information disclosed in the above background art section is only used to strengthen the understanding of the background of the present disclosure, and thus may include information that does not constitute the prior art known to those of ordinary skill in the art. Summary of the Invention

[0008] The object of the present invention is to provide a method for detecting the purity of modified nucleoside monomers and separating key impurities by reversed-phase high-performance liquid chromatography, which can efficiently and accurately detect the purity of modified nucleoside monomers, the residual amounts of process impurities and degradation impurities, and ensure the effective evaluation of the purity of modified nucleoside monomers, thereby well controlling the quality of modified nucleoside monomers.

[0009] To achieve the above object, the present invention provides a method for detecting the purity of modified nucleoside monomers and separating key impurities by reversed-phase high performance liquid chromatography. The method includes: using a reversed-phase C18 chromatographic column filled with octadecylsilyl-bonded silica gel or modified octadecylsilyl-bonded silica gel, with a detection wavelength of 260 nm, mobile phase A being 10 mM ammonium acetate aqueous solution with pH = 7.0, mobile phase B being acetonitrile solution, a flow rate of 0.8 mL / min, a column temperature of 25°C, a sample running time of 35 min, and a gradient elution being selected for the elution program; the elution program is as follows: at time 0 min, mobile phase A is 39% and mobile phase B is 61%; at time 20 min, mobile phase A is 5% and mobile phase B is 95%; at time 25 min, mobile phase A is 5% and mobile phase B is 95%; at time 28 min, mobile phase A is 39% and mobile phase B is 61%; at time 35 min, mobile phase A is 39% and mobile phase B is 61%; successively injecting: blank solution, 0.001 mg / mL LOD solution, 0.002 mg / mL LOQ solution, standard resolution solution, reference solution, impurity 1 stock solution, impurity 2 stock solution, impurity 3 stock solution, and test solution. Among them, impurity 1, impurity 2, and impurity 3 are any three impurities among the key impurities; the key impurities include: unreacted raw materials, oxidation impurities, hydrolysis impurities, impurities with two DMTs on the active site OH, and impurities with phosphorus reagents at two sites; for modified nucleoside monomers with a modified group on the base, the key impurities also include: impurities obtained by removing the modified group from the base of the modified nucleoside monomer; among them, the blank solution is acetonitrile; the LOD solution, LOQ solution, standard resolution solution, reference solution, impurity 1 stock solution, impurity 2 stock solution, impurity 3 stock solution, and test solution are all prepared using acetonitrile as the solvent; the reference solution is prepared using a reference substance of modified nucleoside monomer, with a concentration of 2.0 mg / mL; the concentration of the reference substance in the LOD solution is 0.001 mg / mL, and the concentration of the reference substance in the LOQ solution is 0.002 mg / mL; the impurity 1 stock solution, impurity 2 stock solution, and impurity 3 stock solution are prepared by mixing three different impurities in the synthesis of modified nucleoside monomers, and the concentration of each impurity stock solution is 0.2 mg / mL; in the standard resolution solution, the concentration percentage of each impurity is 0.1%, and the concentration of the reference substance is 2.0 mg / mL; the test solution is prepared using a sample from the synthesis of modified nucleoside monomers, with a concentration of 2.0 mg / mL.

[0010] The detection wavelength of the present invention is 260 nm. At this detection wavelength, interference peaks can be reduced, which is the optimal detection wavelength screened by the inventor. At other wavelengths (such as 235 nm), some interference peaks will appear. The column temperature of the present invention is selected as room temperature. If the temperature is too high, the sample may deteriorate, which will affect the judgment of impurities and thus the analysis of impurities. The flow rate of the present invention is 0.8 mL / min. If the flow rate is lower than 0.8 mL / min, it will not only cause a longer time but also result in a poor peak shape. In addition, the setting of the gradient elution program of the present invention can prevent impurities from remaining in the column and can elute the impurities well.

[0011] Preferably, the method should meet the following conditions: the number of injection needles of the blank solution is at least 2, and there are no obvious impurity peaks at the peak position of the sample; the number of injection needles of the LOD solution is 1, and the signal-to-noise ratio S / N ≥ 3.0; the number of injection needles of the LOQ solution is 1, and the signal-to-noise ratio S / N ≥ 10.0; the number of injection needles of the standard resolution solution is 6, the RSD of the retention time of the main peaks of the 6 needles ≤ 3.0%, the RSD of the peak areas of the main peaks of the 6 needles ≤ 3.0%, the resolution of the key impurities and the main peak side ≥ 1.50; the number of injection needles of the reference solution is 6, the RSD of the retention time of the main peaks of the 6 needles ≤ 3.0%, the RSD of the peak areas of the main peaks of the 6 needles ≤ 3.0%; the number of injection needles of the impurity 1 stock solution, impurity 2 stock solution and impurity 3 stock solution are all 1, the difference in retention time between the main peak and the resolution solution shall not exceed ±0.2 min, and the impurity recovery rate is between 40% and 160%; the number of injection needles of the test solution is 1, and the difference in retention time between the main peak and the resolution solution shall not exceed ±0.2 min.

[0012] Preferably, the reversed-phase C18 chromatographic column is selected from Gemini 3μm NX-C18 or Megassil Cloak C18-B.

[0013] Preferably, in the reversed-phase C18 chromatographic column, the particle size of the packing is 3 μm, the column length is 250 mm, and the inner diameter is 4.6 mm. The chromatographic column selected in the present invention has a long column length, which is beneficial to the separation of impurities, and the particle size of the packing is moderate, which will not cause sample residue.

[0014] Preferably, the injection volume is 5.0 μL; and / or, the needle washing water is water and acetonitrile with a volume ratio of 7:3.

[0015] Preferably, the detector is selected from Agilent PDA detector, DAD detector or TUV detector; the injector is selected from Agilent automatic injector; the test instrument is selected from Agilent 1260 high performance liquid chromatograph.

[0016] Preferably, the modified nucleoside monomers include: fluorine-containing modified nucleoside monomers, methoxy-modified nucleoside monomers, methoxyethyl-modified nucleoside monomers, and tert-butyldimethylsilyl-modified nucleoside monomers.

[0017] Preferably, the modified nucleoside monomers include modified nucleoside monomers with the following chemical structural formulas: Formula I Wherein, R is selected from F, OMe, OMOE, OTBDMS and other modifying groups; Base is selected from adenine base, guanine base, cytosine base, uracil base or their modified bases.

[0018] Preferably, the impurities include the following chemical structures: ; ; Ia; Ib; ; ; Ic; Id; ; ; Ie; If; ; ; Ig; IIc Most of the above impurities are due to the unstable nature of the final product, and it is relatively easy to produce hydrolysis and oxidation impurities during the last step of preparation. Among them, Ia is the unreacted raw material; Ib is the oxidation impurity; Id, Ie and Ig are the hydrolyzed impurities; Ic is due to the relatively similar activities of two sites during the DMT addition process, so there may be a situation where two are added; If is due to the possible dropping of DMT when adding the phosphorus reagent, and at this time the phosphorus reagent may also be added to this site; IIc is an impurity in which the modifying group is removed from the base of the modified nucleoside monomer for nucleoside monomers with modified bases. When separating impurities, hydrolysis and oxidation impurities, as well as unreacted raw materials, are preferentially considered.

[0019] Preferably, the test solution is selected from samples of modified nucleoside monomers synthesized in three different batches.

[0020] The method for detecting the purity of modified nucleoside monomers and separating key impurities by reversed-phase high performance liquid chromatography according to the present invention has the following advantages: The present invention uses a method of reversed-phase high performance liquid chromatography to analyze the purity of modified nucleoside monomers, and also separates and analyzes the key impurities that may be generated during the synthesis process. Factors such as intermediate precision, specificity, detection limit, quantitation limit, and reproducibility are investigated. Finally, the analysis method used has the advantages of good separation effect between impurities and the main peak, good method reproducibility, high intermediate precision, good specificity, and simple operation. This is conducive to better analyzing the purity and impurities of modified nucleoside monomers, and thus helps to achieve the design and synthesis of more complex small nucleic acid molecules.

[0021] The method of the present invention solves the problems that the current pharmacopoeia has no detection method for the purity of nucleoside monomers containing methoxy modification and the separation of key impurities, and cannot effectively monitor the process impurities and degradation impurities of this type of nucleoside monomer. It is extremely suitable for the purity analysis of nucleoside monomers, has a good separation effect on the process impurities and degradation impurities of this compound, fills the blank of the purity detection method for nucleoside monomers, and has high technical value and promotion space. Description of the Drawings

[0022] Figure 1 It is the chromatogram of the blank solution in Example 1 of the present invention.

[0023] Figure 2 It is the chromatogram of the LOD solution in Example 1 of the present invention.

[0024] Figure 3 It is the chromatogram of the LOQ solution in Example 1 of the present invention.

[0025] Figure 4 It is the chromatogram of the impurity 1 stock solution in Example 1 of the present invention.

[0026] Figure 5 It is the chromatogram of the impurity 2 stock solution in Example 1 of the present invention.

[0027] Figure 6 It is the chromatogram of the impurity 3 stock solution in Example 1 of the present invention.

[0028] Figure 7 It is the chromatogram of the resolution solution in Example 1 of the present invention.

[0029] Figure 8 It is the enlarged chromatogram of the resolution solution in Example 1 of the present invention.

[0030] Figure 9 It is the chromatogram of the reference solution in Example 1 of the present invention.

[0031] Figure 10 It is the chromatogram of the test solution 1 in Example 1 of the present invention.

[0032] Figure 11It is the chromatogram of the test sample 2 solution in Example 1 of the present invention.

[0033] Figure 12 It is the chromatogram of the test sample 3 solution in Example 1 of the present invention. Detailed implementation manners

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0035] It should be noted that: for those not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified in the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0036] In the present invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are only for the sake of simplicity and convenience. Accordingly, the description of the numerical range or percentage range should be regarded as having covered and specifically disclosed all possible sub-ranges and individual numerical values within the range (including integers and fractions).

[0037] The features mentioned in the present invention can be combined arbitrarily as long as there is no contradiction in the combination of these features. All possible combinations should be considered as the scope described in this specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0038] The reagents used in the following examples are as follows: (1) Ultra-pure water is used for water, and Watsons or equivalent is selected; acetonitrile (ACN), HPLC grade, and Sigma or equivalent can be selected. After filtering through a 0.22 μm filter membrane, it is used as the sample solvent and also as the blank solution (Blank); ammonium acetate (CH3COONH4), HPLC grade, and Fisher or equivalent can be selected; 2’-F-dA(Bz)-CE-Phosphoramidite is used as the reference substance; three batches of samples produced by the 2’-F-dA(Bz)-CE-Phosphoramidite process are used as the samples to be detected; (2) Preparation of 10 mM ammonium acetate aqueous solution: Weigh 0.77 g of ammonium acetate into a 1000 mL volumetric flask, dissolve it with ultra-pure water, make up the volume to the scale line, mix well by ultrasonic, and then filter through a 0.22 μm filter membrane before use; (3)Preparation of 0.001 mg / mL limit of detection (LOD) solution: Accurately pipette 1 mL of the reference solution into a 100 mL volumetric flask, then add the sample solvent to the calibration line and shake well; then pipette 1 mL of the above solution into a 20 mL volumetric flask, and then add the sample solvent to the calibration line and shake well; (4)Preparation of 0.002 mg / mL limit of quantitation (LOQ) solution: Accurately pipette 1 mL of the reference solution into a 100 mL volumetric flask, then add the sample solvent to the calibration line and shake well; then pipette 1 mL of the above solution into a 10 mL volumetric flask, and then add the sample solvent to the calibration line and shake well; (5)Preparation of 0.2 mg / mL impurity stock solution: Accurately weigh 10.0 ± 0.3 mg of impurity 1, impurity 2, and impurity 3 into a 5 mL volumetric flask respectively, add the sample solvent to dissolve, then make up to the calibration line and shake well; respectively pipette 1 mL of the above solution into a 10 mL volumetric flask, then add the sample solvent to the calibration line and shake well, and record them as impurity 1 stock solution, impurity 2 stock solution, and impurity 3 stock solution respectively; (6)Preparation of standard resolution solution with impurity concentration percentage (i.e., the percentage of impurity mass to the reference mass) of 0.1%: Accurately weigh 20 ± 0.3 mg of the reference into a 10 mL volumetric flask, then accurately pipette 100 μL of the above impurity stock solution into the same volumetric flask respectively, then add the sample solvent to dissolve and make up to the calibration line and shake well; (7)Preparation of 2.0 mg / mL reference solution (STD): Accurately weigh 20 ± 0.3 mg of the reference into a 10 mL volumetric flask, add the sample solvent to dissolve, then make up to the calibration line and shake well; (8)Preparation of 2.0 mg / mL test solution: Accurately weigh 10.0 ± 0.3 mg of the test samples of different batches into 5 mL volumetric flasks respectively, add the sample solvent to dissolve, then make up to the calibration line and shake well to obtain test solutions of different batches, and record them as test solution 1, test solution 2, and test solution 3 respectively.

[0039] Example 1: A method for detecting the purity of modified nucleoside monomers and separating key impurities by reversed-phase high-performance liquid chromatography. In this example, for fluorine-containing modified nucleoside monomers, taking 2’-F-dA(Bz)-CE-Phosphoramidite (where R is F and Base is a base modified with benzoyl group in formula I) as an example, its structural formula is shown as formula II below: ; Formula II ; Formula I Verification and analysis were carried out on Impurity 1 (raw material without phosphorus reagent, Formula IIa), Impurity 2 (phosphorus oxidation impurity, Formula IIb), and Impurity 3 (impurity with benzoyl group removed from the base, Formula IIc) that may be generated during the production process of 2'-F-dA(Bz)-CE-Phosphoramidite, as follows: ; ; ; Formula IIa Formula IIb Formula IIc Three batches of samples obtained from the production process of 2'-F-dA(Bz)-CE-Phosphoramidite were used as samples to be detected. The chromatographic column was specifically selected as a reverse-phase C18 chromatographic column (Gemini 3μm NX-C18 110Å, particle size 3μm, 250 (L) × 4.6 (ID) mm), the detection wavelength was 260 nm, the injection volume was 5.0 μL, mobile phase A (MPA) was 10 mM ammonium acetate aqueous solution with pH = 7.0, mobile phase B (MPB) was acetonitrile solution, the flow rate of the solution in the chromatographic column was 0.8 mL / min, the column temperature was room temperature (25°C), the sample running time was 35 min, the needle washing solution was in a volume ratio of 7:3 of water to acetonitrile, and the elution program was a gradient elution, as shown in Table 1.

[0040] The test instrument was an Agilent 1260 high-performance liquid chromatograph, the detector was an Agilent PDA detector, and the injector was an Agilent automatic injector.

[0041] Table 1 shows the gradient elution conditions of the reverse-phase high-performance liquid chromatography method in Example 1 of the present invention.

[0042] Injections were carried out in sequence: blank solution (acetonitrile), 0.001mg / mL LOD solution, 0.002mg / mL LOQ solution, resolution solution, STD solution, Impurity 1 stock solution, Impurity 2 stock solution, Impurity 3 stock solution, test sample 1 solution, test sample 2 solution, test sample 3 solution.

[0043] The verification requirements and results are shown in Table 2 below and Figures 1 - 12 as follows.

[0044] Table 2 shows the verification requirements and results of the reverse-phase high-performance liquid chromatography.

[0045] The above RSD value was calculated according to the following formula (1): (1) In formula (1),n For the number of measurements, n = 6 in the present invention; x It can be the retention time of the main peak or the peak area of the main peak as needed; x i It is the value measured each time in 6 groups of repeated experiments; It is the average value calculated in 6 groups of repeated experiments.

[0046] The above recovery rate is calculated according to the following formula (1): (2) In formula (2), A res It is the peak area of a single impurity in the resolution solution; A std It is the peak area of a single impurity in the reference solution; A impurity It is the peak area of a single impurity in the impurity stock solution; 100 is the dilution factor of the impurity.

[0047] Ensure that there is no obvious interference from impurity peaks at the elution position of the main peak in the blank solution. The corresponding chromatogram of the blank solution is as Figure 1 shown; the corresponding chromatogram of the LOD solution is as Figure 2 shown, and the corresponding chromatogram of the LOQ solution is as Figure 3 shown; the corresponding chromatograms of the stock solutions of impurity 1, impurity 2, and impurity 3 are respectively Figure 4 , Figure 5 and Figure 6 ; the corresponding chromatogram of the standard resolution solution is as Figure 7 shown. The enlarged view of the resolution solution is as Figure 8 shown. The standard resolution solution can better reflect the separation situation between the three key impurities and the product main peak; the corresponding chromatogram of the reference solution is as Figure 9 shown; the corresponding chromatograms of the test solutions 1, test solutions 2, and test solutions 3 of three batches are respectively as Figure 10 , Figure 11 and Figure 12 shown.

[0048] The elution time of the corresponding impurity and the main peak in the resolution solution and their separation situation are shown in Table 3. It can be seen from the chromatogram and the following table that the product main peak and the three key impurities can be completely separated, meeting the separation requirements.

[0049] Table 3 Impurity separation situation corresponding to the resolution solution

[0050] From the analysis of the data in the above table, it can be concluded that the analytical method involved in the present invention can well separate the key impurities that may be generated in the production of 2’-F-dA(Bz)-CE-Phosphoramidite process, and the resolution of this method is relatively high and the separation effect is good. By changing some key factors in the analytical method, such as the selection of the mobile phase, the mobile phase ratio, the column temperature, the flow rate, and the method gradient, the best separation effect can be achieved, and the purity of nucleic acid monomers can be evaluated efficiently and accurately.

[0051] It should be noted that the retention times of the main peaks of the samples measured by two equivalent instruments shall not exceed ±1.0 min.

[0052] The test instrument can also be an instrument equivalent to the Agilent 1260 high performance liquid chromatograph. In addition to the PDA detector, the DAD detector or the TUV detector or an equivalent instrument can also be selected.

[0053] In addition, for fluorine-modified nucleoside monomers, they are not limited to the fluorine-modified nucleoside monomers shown in the following structural formulas: ; ; ;

[0054] Comparative Example 1: Referring to the method of Example 1, keeping other conditions unchanged, only changing the type of chromatographic column to detect the purity and key impurities of the modified nucleoside monomer. The chromatographic column selected in this comparative example is Welch Xtimate ® C18, 4.6 (ID) × 150 (L) mm, packing particle size 3μm, and the resolution data obtained are shown in Table 4.

[0055] Table 4 Separation of impurities in the solution corresponding to the resolution obtained by changing the type of chromatographic column

[0056] Referring to Table 4, it can be seen that the resolution between impurity 2 and impurity 3 is relatively poor compared with the resolution in Example 1, and the required separation requirement is not achieved.

[0057] Comparative Example 2: Referring to the method of Example 1, keeping other conditions unchanged, only changing the column temperature to detect the purity and key impurities of the modified nucleoside monomer. The column temperature selected in this comparative example is 40°C, and the resolution data obtained are shown in Table 5.

[0058] Table 5 Separation of impurities in the solution corresponding to the resolution obtained by changing the column temperature

[0059] Referring to Table 5, it can be seen that although the resolution between Impurity 2 and Impurity 3 meets the required separation requirements, compared with Example 1, the separation effect is not as good as that in Example 1.

[0060] In summary, the method of the present invention has the advantages of good separation effect, simple operation, high safety factor, etc. It solves the current disadvantages that due to the characteristics of modified nucleoside monomers such as difficult vaporization and high boiling point, it is impossible to detect their purity by gas chromatography method, and also solves the disadvantage that the existing analysis methods cannot well separate the impurities generated in the process of modified nucleoside monomer production. The reverse-phase high-performance liquid chromatography method adopted in the present invention not only verifies the analysis method of modified nucleoside monomers, but also separates and analyzes the key impurities that may be generated in the synthesis process, and examines factors such as intermediate precision, specificity, detection limit, quantitation limit and reproducibility. Finally, the method used has the advantages of good separation effect between impurities and the main peak, good method reproducibility, high intermediate precision, good specificity, simple operation, etc. This is conducive to better analysis of the purity and impurities of modified nucleoside monomers, and thus helps to realize the design and synthesis of more complex small nucleic acid molecules.

[0061] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions of the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. A method for detecting the purity of modified nucleoside monomers and separating key impurities by reversed-phase high performance liquid chromatography, characterized in that, The method includes: Using a reversed-phase C18 chromatographic column packed with octadecylsilyl-bonded silica gel or modified octadecylsilyl-bonded silica gel as the filler, with a detection wavelength of 260 nm, mobile phase A being a 10 mM aqueous ammonium acetate solution with pH = 7.0, mobile phase B being an acetonitrile solution, a flow rate of 0.8 mL / min, a column temperature of 25 °C, a sample running time of 35 min, and a gradient elution program being selected for the elution procedure; The elution procedure is as follows: At 0 min, mobile phase A is 39% and mobile phase B is 61%; At 20 min, mobile phase A is 5% and mobile phase B is 95%; At 25 min, mobile phase A is 5% and mobile phase B is 95%; At 28 min, mobile phase A is 39% and mobile phase B is 61%; At 35 min, mobile phase A is 39% and mobile phase B is 61%; Inject samples in sequence: blank solution, 0.001 mg / mL LOD solution, 0.002 mg / mL LOQ solution, standard resolution solution, reference solution, impurity 1 stock solution, impurity 2 stock solution, impurity 3 stock solution, and test solution; among them, impurity 1, impurity 2, and impurity 3 are any three impurities among the key impurities; The key impurities include: unreacted raw materials, oxidation impurities, hydrolysis impurities, impurities with two DMTs on the active site OH, and impurities with phosphorus reagents at two sites; for modified nucleoside monomers with modified groups on the base, the key impurities also include: impurities with the modified group removed from the base of the modified nucleoside monomer; Among them, the blank solution is acetonitrile; the LOD solution, LOQ solution, standard resolution solution, reference solution, impurity 1 stock solution, impurity 2 stock solution, impurity 3 stock solution, and test solution are all prepared using acetonitrile as the solvent; the reference solution is prepared using a modified nucleoside monomer reference substance with a concentration of 2.0 mg / mL; the concentration of the reference substance in the LOD solution is 0.001 mg / mL, and the concentration of the reference substance in the LOQ solution is 0.002 mg / mL; the impurity 1 stock solution, impurity 2 stock solution, and impurity 3 stock solution are prepared by formulating three different impurities in the synthesis of modified nucleoside monomers, and the concentration of each impurity stock solution is 0.2 mg / mL; in the standard resolution solution, the concentration percentage of each impurity is 0.1%, and the concentration of the reference substance is 2.0 mg / mL; the test solution is prepared using a sample synthesized from modified nucleoside monomers with a concentration of 2.0 mg / mL.

2. The method according to claim 1, characterized in that, This method should meet the following conditions: The number of injection needles for the blank solution is at least 2 needles, and there are no obvious impurity peaks at the sample peak position; The number of injection needles for the LOD solution is 1 needle, and the signal-to-noise ratio S / N ≥ 3.0; The number of injection needles for the LOQ solution is 1 needle, and the signal-to-noise ratio S / N ≥ 10.0; The number of injection needles for the standard resolution solution is 6 needles, the RSD of the retention time of the main peaks of the 6 needles ≤ 3.0%, the RSD of the peak areas of the main peaks of the 6 needles ≤ 3.0%, and the resolution of the key impurities and the peaks beside the main peak ≥ 1.50; The reference solution is injected 6 times, and the RSD of the retention times of the main peaks of the 6 injections ≤ 3.0%, and the RSD of the peak areas of the main peaks of the 6 injections ≤ 3.0%; The impurity 1 stock solution, impurity 2 stock solution, and impurity 3 stock solution are each injected 1 time. The difference in the retention time between the main peak and the resolution solution shall not exceed ±0.2 min, and the impurity recovery rate is between 40% and 160%; The test solution is injected 1 time. The difference in the retention time between the main peak and the resolution solution shall not exceed ±0.2 min.

3. The method according to claim 1, wherein The reverse-phase C18 chromatographic column is selected from Gemini 3μm NX-C18 or Megassil Cloak C18-B.

4. The method according to claim 1, characterized in that, In the reverse-phase C18 chromatographic column, the particle size of the packing is 3 μm, the column length is 250 mm, and the inner diameter is 4.6 mm.

5. The method according to claim 1, wherein The injection volume is 5.0 μL; and / or, the needle washing solution is water and acetonitrile with a volume ratio of 7:

3.

6. The method according to claim 1, wherein The detector is selected from Agilent PDA detector, DAD detector, or TUV detector; the injector is selected from Agilent autosampler; the test instrument is selected from Agilent 1260 high performance liquid chromatograph.

7. The method according to claim 1, wherein The modified nucleoside monomers include: fluorine-containing modified nucleoside monomers, methoxy-modified nucleoside monomers, methoxyethyl-modified nucleoside monomers, and tert-butyldimethylsilyl-modified nucleoside monomers.

8. The method according to claim 7, characterized in that, The chemical structure of the modified nucleoside monomer is shown in Formula I: ； Formula I; Wherein, R is selected from F, OMe, OMOE, or OTBDMS; Base is selected from adenine base, guanine base, cytosine base, uracil base, or their modified bases.

9. The method according to claim 8, wherein The impurities include the following chemical structures: ； ； Ia; Ib; ； ； Ic; Id; ； ； Ie; If; ； ； Ig; IIc.

10. The method according to any one of claims 1 to 9, characterized in that The test solution is selected from modified nucleoside monomer samples synthesized from three different batches.

Images