A rapid liquid chromatography analysis method for phosphoramidite monomers

Abstract

The present invention discloses a rapid liquid chromatography analysis method for phosphoramidite monomers, comprising the following steps: preparing a sample solution: weighing a sample and placing it in a volumetric flask, dissolving and then constant volume to obtain a sample solution; preparing mobile phase A and mobile phase B, filtering through a microporous membrane, and ultrasonically degassing the sample solution, and then detecting the phosphoramidite purity using reversed-phase high-performance liquid chromatography; and calculating the purity results using an area normalization method. The present invention adopts reversed-phase high-performance liquid chromatography for analysis, which has the advantages of short analysis time, good component peak separation effect, high resolution, high analytical accuracy, good reproducibility, and simple operation, thereby achieving effective control of the quality of the phosphoramidite.

Description

Technical Field

[0001] The invention belongs to the field of analysis and relates to a rapid liquid chromatography analysis method for phosphoramidite monomers. Background Art

[0002] Oligonucleotides are a general term for short nucleotide chains consisting of approximately 20 bases (including nucleotides in deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Oligonucleotides can easily bind to their complementary chains through the principle of base pairing. With their unique mechanism of action, they represent a new field of disease treatment and have been a hot topic in pharmaceutical research for the past decade. Small nucleic acid drugs include antisense oligonucleotides (ASOs), immunostimulatory oligonucleotides, ribozymes, mirconaviruses (mircoRNAs), siRNAs, aptamers, and mirror oligonucleotides.

[0003] Phosphoramidites are essential for the synthesis of small nucleic acid APIs (oligonucleotides), primarily DNA and RNA derivatives, and their derivatives. The timely supply of quality-compliant nucleoside monomers is crucial for the commercialization of small nucleic acid drugs. Chemical modification techniques during API synthesis and delivery technologies during formulation are crucial for the success of small nucleic acid drugs.

[0004] Therefore, the purity of the important phosphoramidite monomer directly affects the quality of downstream products.

[0005] Because phosphoramidite monomers have high boiling points and are difficult to vaporize, gas chromatography is not suitable for determining their purity. Furthermore, there are currently no reports on methods for analyzing phosphoramidite purity. Summary of the Invention

[0006] In view of the lack of research on existing phosphoramidite purity analysis methods, the present invention aims to provide a high performance liquid chromatography analysis method for phosphoramidite monomers. The present invention adopts reversed-phase high performance liquid chromatography for analysis, which has the advantages of short analysis time, good component peak separation effect, high resolution, high analysis accuracy, good reproducibility, simple operation, etc., so as to achieve effective control of the quality of phosphoramidites.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A rapid liquid chromatography analysis method for phosphoramidite monomers, comprising the following steps:

[0009] 1) Prepare sample solution: weigh the sample and place it in a volumetric flask, dissolve it and adjust the volume to obtain the sample solution;

[0010] 2) preparing mobile phase A and mobile phase B, filtering through a microporous membrane, ultrasonically degassing, and then testing the sample solution by reverse-phase high performance liquid chromatography to determine the purity of the phosphoramidite;

[0011] 3) The area normalization method was used to calculate the purity results.

[0012] As a preferred embodiment of the present invention, in step 1), the concentration of the sample solution is 0.5-1.0 mg / mL, and the solvent used for dissolution is acetonitrile.

[0013] As a preferred embodiment of the present invention, in step 2), the reverse-phase high performance liquid chromatography uses a Waters Priier high performance liquid chromatograph equipped with an ultraviolet detector and full wavelength detection.

[0014] As a preferred embodiment of the present invention, in step 2), the chromatographic column is a Water Acquily BEH C18 chromatographic column, and the chromatographic column specifications are a length of 100 mm, an inner diameter of 2.1 mm, and a filler particle size of 1.7 μm.

[0015] As a preferred embodiment of the present invention, in step 2), mobile phase A is a 10 mM ammonium acetate aqueous solution, and mobile phase B is chromatographic grade acetonitrile;

[0016] The elution gradient was: 0 min, 40% mobile phase A: 60% mobile phase B; 12 min, 10% mobile phase A: 90% mobile phase B; 12.1 min, 40% mobile phase A: 60% mobile phase B; 15 min, 40% mobile phase A: 60% mobile phase B.

[0017] As a preferred embodiment of the present invention, in step 2), the detection conditions are: detection wavelength 235 nm; flow rate 0.3 mL / min; injection volume 2 μL; column temperature 30° C.; and injection plate temperature 10° C.

[0018] As a preferred embodiment of the present invention, in step 2), the microporous membrane used for filtration is 0.22 μm.

[0019] As a preferred embodiment of the present invention, in step 2), the ultrasonic degassing time is 15 minutes.

[0020] The present invention adopts the above technical solution and has the following beneficial effects:

[0021] (1) The present invention has a short analysis time. Using the method of the present invention for detection, it only takes 20 minutes from sample pretreatment to completion of result analysis;

[0022] (2) The component peak separation effect is good, with a theoretical plate number of up to 30,000, which can completely separate the components contained in the sample.

[0023] (3) High resolution. The best separation effect can be achieved by selecting the type and ratio of the stationary phase and mobile phase.

[0024] (4) High analytical precision.

[0025] (5) Good reproducibility, small error, and stable analysis results.

[0026] (6) The operation is simple, the analysis is fully automated, and the detection is completed by the instrument. Regardless of whether you have experimental knowledge or not, you can operate it after receiving operation training.

[0027] (7) Achieve effective control of the quality of phosphoramidite and purity test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : The results of DMT-dA(Bz)-CE-phosphoramidite analysis of samples.

[0029] Figure 2 : The results of DMT-dT-CE-phosphoramidite analysis of sample detection.

[0030] Figure 3 : The results of DMT-dC(Bz)-CE-phosphoramidite analysis of sample detection.

[0031] Figure 4 : The results of DMT-dG(iBu)-CE-phosphoramidite analysis of sample detection. DETAILED DESCRIPTION

[0032] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further described below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the embodiments, other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0033] In the present invention, the instruments used include:

[0034] (1) Waters Priier high performance liquid chromatograph with UV detector and full wavelength detection;

[0035] (2) Chromatographic column: Water Acquily BEH C18 (100×2.1mm, 1.7μm);

[0036] (3) Ultrasonic degassing device.

[0037] The present invention provides a rapid liquid chromatography analysis method for phosphoramidite monomers, including instrument selection, setting of detection conditions, sample processing, and detection;

[0038] The detection method of the present invention is used to detect the purity of phosphoramidite in less than 15 minutes.

[0039] The setting of the detection conditions and the volume ratio of the elution gradient are shown in Table 1;

[0040] Table 1. Elution gradient

[0041] Time(min) A％ B％ 0 40 60 12 10 90 12.1 40 60 15 40 60

[0042] Mobile phase A was 10 mM NH4OAc in H2O; mobile phase B was chromatographic grade acetonitrile.

[0043] Diluent / Blank: Acetonitrile.

[0044] The detection conditions were set as follows: detection wavelength 235 nm, flow rate 0.3 mL / min, injection volume 2 μL, column temperature 30°C, and injection plate temperature 10°C.

[0045] Example 1

[0046] The rapid liquid chromatography analysis method for phosphoramidite monomers provided in this embodiment includes:

[0047] Sample processing:

[0048] 1) Accurately weigh 6 mg of sample into a 10 ml volumetric flask, add a small amount of acetonitrile to dissolve and then make up to volume.

[0049] 2) The prepared mobile phase was first filtered through a 0.22 μm membrane, then ultrasonically degassed for 15 minutes, and then placed on a high performance liquid chromatograph for degassed use.

[0050] 3) After the peak appears, use the area normalization method to calculate the purity results.

[0051] See attached figure, Figure 1 The results of DMT-dA(Bz)-CE-phosphoramidite analysis were obtained, with a main peak resolution of 4.1 and a theoretical plate number of 40,000. Figure 2 The results of DMT-dT-CE-phosphoramidite analysis showed a main peak resolution of 5.1 and a theoretical plate number of 30,000. Figure 3The results of DMT-dC(Bz)-CE-phosphoramidite analysis of samples were obtained, with a main peak resolution of 5.4 and a theoretical plate number of 40,000. Figure 4 The results of DMT-dG(iBu)-CE-phosphoramidite analysis of the sample showed a main peak resolution of 4.5 and a theoretical plate number of 30,000. This demonstrates that the present invention provides excellent peak separation, completely separating the components in the sample. High resolution is achieved by selecting the types and ratios of the stationary and mobile phases, which can optimize separation.

[0052] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A rapid liquid chromatography analysis method for phosphoramidite monomers, characterized in that: The phosphoramidite monomer liquid chromatography rapid analysis method comprises the following steps: 1) Prepare sample solution: weigh the sample and place it in a volumetric flask, dissolve it and adjust the volume to obtain the sample solution; 2) preparing mobile phase A and mobile phase B, filtering through a microporous membrane, ultrasonically degassing, and then testing the sample solution by reverse-phase high performance liquid chromatography to determine the purity of the phosphoramidite; 3) Use area normalization method to calculate purity results; In step 2), the reversed-phase high performance liquid chromatography was performed using a Waters Priier high performance liquid chromatograph equipped with a UV detector and full wavelength detection; In step 2), the chromatographic column is a Water Acquily BEH C18 chromatographic column with a length of 100 mm, an inner diameter of 2.1 mm, and a filler particle size of 1.7 μm; In step 2), mobile phase A is 10 mM ammonium acetate aqueous solution, and mobile phase B is chromatography-grade acetonitrile; The elution gradient was as follows: 0 min, 40% mobile phase A: 60% mobile phase B; 12 min, 10% mobile phase A: 90% mobile phase B; 12.1 min, 40% mobile phase A: 60% mobile phase B; 15 min, 40% mobile phase A: 60% mobile phase B; In step 2), the detection conditions are as follows: detection wavelength 235 nm; flow rate 0.3 mL / min; injection volume 2 μL; column temperature 30° C.; injection plate temperature 10° C.; The samples detected by the method are DMT-dA(Bz)-CE-phosphoramidite, DMT-dT-CE-phosphoramidite, DMT-dC(Bz)-CE-phosphoramidite and DMT-d G (iBu) -CE-phosphoramidite.

2. The rapid liquid chromatography analysis method for phosphoramidite monomers according to claim 1, characterized in that: In step 1), the concentration of the sample solution is 0.5-1.0 mg / mL, and the solvent used for dissolution is acetonitrile.

3. The rapid liquid chromatography analysis method for phosphoramidite monomers according to claim 1, characterized in that: In step 2), the microporous membrane used for filtration is 0.22 μm.

4. The rapid liquid chromatography analysis method for phosphoramidite monomers according to claim 1, characterized in that: In step 2), the ultrasonic degassing time is 15 minutes.

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