Method for confirming liquid chromatogram-mass spectrum combined oligonucleotide sequence

Abstract

The invention provides a liquid chromatography-mass spectrometry combined oligonucleotide sequence confirmation method, and relates to the technical field of instrument analysis. The confirmation method comprises the following steps: detecting a sample solution containing oligonucleotide by adopting an IPRP-UPLC-MS / MS method to obtain fragment ion data; and processing the fragment ion data by adopting CONFIRM Sequence to obtain a sequence matching rate. According to the method, IPRP-LC-MS / MS is adopted to detect a sample solution, CONFIRM Sequence is adopted to process data, and the sequence matching rate is obtained. The method disclosed by the invention adopts an IPRP high performance liquid chromatography and QTOF MS combined method for analysis, has the advantages of short analysis time, strong directness, high analysis precision, high resolution, high accuracy, simplicity in operation and the like, and achieves the purpose of identifying the oligonucleotide sequence.

Description

Technical Field

[0001] This invention relates to the field of instrumental analysis technology, and in particular to a method for confirming oligonucleotide sequences using liquid chromatography-mass spectrometry. Background Technology

[0002] Oligonucleotide drugs (such as ASO, siRNA, and aptamer) are short-chain nucleic acid molecules composed of a dozen to several dozen nucleotides. The correctness of their sequence is fundamental to their pharmacological activity, specificity, and safety. Therefore, accurate sequence confirmation after synthesis is a crucial step in drug development and quality control. Sequencing of oligonucleotide drugs primarily aims to confirm whether the synthesized product matches the designed theoretical sequence. This includes detecting sequence impurities such as deletions, insertions, and base mispairing. Due to the high polarity, high charge, ease of modification, and complex structure of oligonucleotides, their sequence analysis must rely on high-resolution, highly selective analytical techniques.

[0003] Mass spectrometry-based oligonucleotide sequencing boasts high sensitivity, high resolution, and direct structure resolution capabilities. It enables sequence confirmation via fragment ions and accurately identifies base deletions, modifications, and degradation impurities, making it the most reliable sequence confirmation method currently available. Compared to traditional indirect methods such as enzymatic digestion and electrophoresis, MS-based sequencing can simultaneously obtain molecular weight, structure, and purity information in a single analysis, offering significant advantages such as high throughput, strong quantification, and intuitive results. However, this technology also presents challenges, including complex data analysis and the large volume of fragment maps.

[0004] Despite the complexity of nucleotide mass spectrometry fragmentation, the widespread adoption of LC-MS / MS technology in oligonucleotide sequencing is due to the continuous development of bioinformatics tools that can facilitate data analysis and increase throughput. This invention utilizes UPLC-MS / MS (QTOF MS) technology and the CONFIRM Sequence application from Waters, enabling automated processing of data from targeted MS / MS or non-targeted MS. E MS / MS spectra of oligonucleotides obtained by (DIA) were used to automatically sequence the synthetic oligonucleotides and their impurities.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] This invention provides a method for oligonucleotide sequence confirmation using liquid chromatography-mass spectrometry. The method combines high-resolution mass spectrometry with intelligent software analysis to confirm oligonucleotide sequences, solving the problems of insufficient research on oligonucleotide sequence confirmation analysis methods in the prior art, and the problems of complex data processing and low confirmation efficiency of traditional methods.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for confirming oligonucleotide sequences using liquid chromatography-mass spectrometry, the confirmation method comprising: The sample solution containing oligonucleotides was detected using the IPRP-UPLC-MS / MS method to obtain fragment ion data; The fragment ion data were processed using CONFIRM Sequence to obtain the sequence matching rate.

[0008] Furthermore, the sample solution containing the oligonucleotide drug is prepared by the following steps: The oligonucleotide to be tested is dissolved in water to obtain the sample solution containing the oligonucleotide.

[0009] Furthermore, the concentration of the oligonucleotide-containing sample solution is 0.1~1.0 mg / mL, preferably 0.2~1.0 mg / mL.

[0010] Furthermore, the liquid chromatography used in the detection was a Waters Premier ultra-high performance liquid chromatograph; the mass spectrometry used in the detection was a Waters Xevo G3 high-performance time-of-flight tandem mass spectrometer.

[0011] Furthermore, the chromatographic column used for the detection is an OST BHE C18 column.

[0012] Furthermore, the chromatographic column used for the detection can also be other C18 columns equivalent to the OST BHE C18 column, such as Waters' ACQUITY Premier or ACQUITY UPLC column.

[0013] Furthermore, the specifications of the chromatographic column used for the detection include: a column length of 50~150 mm, an inner diameter of 1.5~4.6 mm, and a packing particle size of 1.5~5.0 μm.

[0014] Furthermore, the chromatographic column used for the detection is a Waters Acquity Premier OST BEH C18 column with a length of 100 mm, an inner diameter of 2.1 mm, and a packing particle size of 3.5 μm.

[0015] Furthermore, in the detection, the mobile phase A used in the liquid chromatography includes dibutylamine acetate, acetonitrile, and water, and the mobile phase B includes dibutylamine acetate, acetonitrile, and water.

[0016] Furthermore, in the mobile phase A, the concentration of dibutylamine acetate is 5-15 mM, the volume percentage of acetonitrile is 5-15%, and the balance is water.

[0017] Furthermore, in the mobile phase B, the concentration of dibutylamine acetate is 5-15 mM, the volume percentage of acetonitrile is 75-90%, and the balance is water.

[0018] Furthermore, the pH of mobile phase A and mobile phase B are each independently 7.0 to 8.0.

[0019] Furthermore, the gradient elution conditions used in the liquid chromatography for the detection are as follows: 0 min, mobile phase A: 85~95%, mobile phase B: 5~15%; 2 min, mobile phase A: 85~95%, mobile phase B: 5~15%; 12 min, mobile phase A: 5~15%; mobile phase B: 85~95%; 12.1 min, mobile phase A: 85~95%; mobile phase B: 5~15%; 15 min, mobile phase A: 85~95% : mobile phase B: 5~15%.

[0020] Furthermore, the gradient elution conditions used in the liquid chromatography for the detection are as follows: 0 min, mobile phase A: 90%, mobile phase B: 10%; 2 min, mobile phase A: 90%, mobile phase B: 10%; 12 min, mobile phase A: 10% : mobile phase B: 90%; 12.1 min, mobile phase A: 90%; mobile phase B: 10%; 15 min, mobile phase A: 90% : mobile phase B: 10%.

[0021] Furthermore, the conditions used in the liquid chromatography for the detection include: wavelength of 250~270 nm; flow rate of 0.1~1.0 mL / min; injection volume of 3~10 μL; column temperature of 35~70℃; and injection plate temperature of 2~10℃.

[0022] Furthermore, the mass spectrometry conditions used in the detection include: ionization mode: ESI; scanning mode: negative polarity; analysis mode: sensitivity mode; precursor ion: 1782.06 m / z; capillary voltage: 1.5~2.5 kV; sample cone voltage: 40~100 V; cone gas flow rate: 45~55 L / h; desolvation gas flow rate: 700~900 L / h; ion source temperature: 100~150℃; desolvation temperature: 300~450℃; mass range: 500~3000 m / z; collision energy voltage: 10~50 V; scan time: 0.5~2 s.

[0023] Furthermore, after preparing the mobile phase A and mobile phase B, the following post-processing steps are required; Mobile phase A and mobile phase B were filtered through a microporous membrane and then degassed by ultrasonication.

[0024] Furthermore, the pore size of the microporous filter membrane is 0.20~0.25 μm.

[0025] Furthermore, the ultrasonic degassing power is 400~600 W, the frequency is 40~60 kHz, and the ultrasonic degassing time is 5~30 min.

[0026] Compared with the prior art, the present invention has the following beneficial effects: (1) The method described in this invention has a wide detection range and can detect different types of oligonucleotide samples. It can effectively analyze a variety of oligonucleotide samples containing different lengths, sequences, chemical modifications and impurities, and the optimal parameters can be selected by adjusting the parameters. At the same time, the key parameters of this method (such as elution gradient, mass spectrometry parameters collision energy voltage, etc.) can be flexibly adjusted and optimized according to the characteristics of specific samples, so as to quickly obtain the optimal detection conditions for specific analytical targets, which significantly improves the practical value and detection efficiency of the method; (2) The method described in this invention is highly direct, obtaining fragment information through LC-MS / MS to directly confirm the sequence rather than relying on indirect inference; (3) The method described in this invention has a short processing time, with a detection time of 15 min. The data can be parsed in a few minutes, which greatly improves the data processing efficiency. (4) The method described in this invention has high analytical precision, high resolution, and high accuracy; (5) The method described in this invention is easy to operate, the analysis is fully automated, and the detection is completed by instruments. Regardless of whether one has experimental background, as long as one has been trained, one can operate it. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the sample detection results provided in Embodiment 1 of the present invention.

[0029] Figure 2 The structure of the oligonucleotide sample provided in Example 1 of this invention is shown below. Detailed Implementation

[0030] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0031] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] In a first aspect, the present invention provides a method for confirming oligonucleotide sequences using liquid chromatography-mass spectrometry, the confirmation method comprising: The sample solution containing oligonucleotides was detected using the IPRP-UPLC-MS / MS method to obtain fragment ion data; The fragment ion data were processed using CONFIRM Sequence to obtain the sequence matching rate.

[0034] In this invention, the sample solution is first detected using IPRP-UPLC-MS / MS (ion-pair reversed-phase ultra-high performance liquid chromatography-tandem mass spectrometry). Collision-induced dissociation (CID) is performed on selected precursor ions to obtain fragment ion spectra. The fragment data is then imported into the CONFIRM Sequence application from Waters, where the software automatically compares the data with established theoretical sequences, identifies the a / b / c / d and w / x / y / z ion series, and generates sequence matching rates. This method is an oligonucleotide sequence identification method combining high-resolution mass spectrometry and intelligent software analysis. It offers advantages such as ease of operation, high accuracy, and rapid result acquisition, achieving the goal of oligonucleotide sequence identification.

[0035] As an optional implementation, the sample solution containing the oligonucleotide drug is prepared by the following steps: dissolving the oligonucleotide to be tested in water to obtain the sample solution containing the oligonucleotide; As an optional implementation, the concentration of the oligonucleotide-containing sample solution is 0.1~1.0 mg / mL, for example, it can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, etc.

[0036] As an optional implementation, the detection is performed using a Waters Premier ultra-high performance liquid chromatograph.

[0037] As an optional implementation, the liquid chromatograph is equipped with an ultraviolet detector and full-wavelength detection.

[0038] As an optional implementation, the mass spectrometry used in the detection is a Waters Xevo G3, a high-performance time-of-flight (Tof) tandem mass spectrometer (MS / MS).

[0039] As an optional implementation, the chromatographic column used for the detection is an OST BHE C18 column.

[0040] As an optional implementation, the specifications of the chromatographic column used for detection include: a column length of 50-150 mm (e.g., 50 mm, 75 mm, 100 mm, 125 mm, 150 mm, etc.), an inner diameter of 1.5-4.6 mm (e.g., 1.5 mm, 2.0 mm, 2.1 mm, 3.0 mm, 4.0 mm, 4.6 mm, etc.), and a packing particle size of 1.5-5.0 μm (e.g., 1.5 μm, 1.7 μm, 1.8 μm, 2.0 μm, 2.5 μm, 2.6 μm, 2.7 μm, 3.0 μm, 3.5 μm, 4.0 μm, 5.0 μm, etc.).

[0041] As a preferred embodiment, the chromatographic column used for the detection is a Waters Acquity Premier OSTBEH C18 column with a length of 100 mm, an inner diameter of 2.1 mm, and a packing particle size of 3.5 μm.

[0042] As an optional implementation, the mobile phase A used in the liquid chromatography of the detection includes dibutylamine acetate, acetonitrile, and water, and the mobile phase B includes dibutylamine acetate, acetonitrile, and water.

[0043] As an optional embodiment, in the mobile phase A, the concentration of dibutylamine acetate is 5-15 mM (e.g., it can be 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, etc.), the volume percentage of acetonitrile is 5-15% (e.g., it can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc.), and the balance is water.

[0044] As an optional implementation, in the mobile phase B, the concentration of dibutylamine acetate is 5-15 mM (e.g., 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, etc.), the volume percentage of acetonitrile is 75-90% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, etc.), and the balance is water.

[0045] As an optional implementation, the pH of each mobile phase A is independently 7.0 to 8.0, for example, it can be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, etc.

[0046] As an optional implementation, the pH of each mobile phase B is independently 7.0 to 8.0, for example, it can be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, etc.

[0047] As an optional implementation, the gradient elution conditions used in the liquid chromatography during the detection are as follows: 0 min, mobile phase A: 85~95% (e.g., it can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, etc.), mobile phase B: 5~15% (e.g., it can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc.). 2 min, mobile phase A: 85~95% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, etc.), mobile phase B: 5~15% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc.). 12 min, mobile phase A: 5~15% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc.); mobile phase B: 85~95% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, etc.). 12.1 min, mobile phase A: 85~95% (e.g., it can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, etc.): mobile phase B: 5~15%; 15 min, mobile phase A: 85~95% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, etc.): mobile phase B: 5~15% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc.). As a preferred embodiment, the gradient elution conditions used in the liquid chromatography during the detection are as follows: 0 min, mobile phase A: 90%, mobile phase B: 10%; 2 min, mobile phase A: 90%, mobile phase B: 10%; 12 min, mobile phase A: 10% : mobile phase B: 90%; 12.1 min, mobile phase A: 90%; mobile phase B: 10%; 15 min, mobile phase A: 90% : mobile phase B: 10%.

[0048] As an optional implementation, the conditions used in the liquid chromatography for the detection include: a wavelength of 250-270 nm (e.g., 250 nm, 252 nm, 254 nm, 256 nm, 258 nm, 260 nm, 262 nm, 264 nm, 268 nm, 270 nm, etc.); a flow rate of 0.1-1.0 mL / min (e.g., 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, etc.); and an injection volume of 3-10 μL (e.g., 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, etc.). μL, etc.); column temperature is 35~70℃ (e.g., 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, etc.); injection plate temperature is 2~10℃ (e.g., 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, 10℃, etc.).

[0049] As an optional implementation, the mass spectrometry conditions used in the detection include: ionization mode: ESI; scan mode: negative polarity; analyzer mode: sensitivity mode; precursor m / z: 1782.06 m / z; capillary voltage: 1.5~2.5 kV (e.g., 1.5 kV, 1.6 kV, 1.7 kV, 1.8 kV, 1.9 kV, 2.0 kV, 2.1 kV, 2.2 kV, 2.3 kV, 2.4 kV, 2.5 kV).5 kV); Sample cone voltage: 40~100 V (e.g., 40 V, 50 V, 60 V, 70 V, 80 V, 90 V, 100 V, etc.); Cone gas flow rate: 45~55 L / h (e.g., 45 L / h, 46 L / h, 47 L / h, 48 L / h, 49 L / h, 50 L / h, 51 L / h, 52 L / h, 53 L / h, 54 L / h, 55 L / h, etc.); Desolvation gas flow rate: 700~900 L / h (e.g., 700 L / h, 720 L / h, 740 L / h, 760 L / h, 780 L / h, 800 L / h, 820 L / h, 840 L / h, 860 L / h, 880 L / h, 900 L / h, etc.). L / h, etc.; Source temperature: 100~150℃ (e.g., 100℃, 105℃, 110℃, 115℃, 120℃, 125℃, 130℃, 135℃, 140℃, 145℃, 150℃, etc.); Desolvation temperature: 300~450℃ (e.g., 300℃, 320℃, 340℃, 360℃, 380℃, 400℃, 420℃, 440℃, 450℃, etc.); Mass range (m / z): 500~3000 m / z (e.g., 500 m / z, 1000 m / z, 1500 m / z, 2000 m / z, 2500 m / z, 3000 m / z, etc.); Collision energy mode: 10~50 V (e.g., 10 V, etc.). The available voltages are V, 15 V, 20 V, 25 V, 30 V, 32 V, 34 V, 36 V, 38 V, 40 V, 42 V, 44 V, 46 V, 48 V, 50 V, etc.; the scan time is 0.5~2 s (e.g., 0.5s, 0.6s, 0.7s, 0.8s, 0.9s, 1.0s, 1.1s, 1.2s, 1.3s, 1.4s, 1.5s, 1.6s, 1.7s, 1.8s, 1.9s, 2.0s, etc.).

[0050] As an optional implementation, the following post-processing steps are required after preparing the mobile phase A and mobile phase B; Mobile phase A and mobile phase B were filtered through a microporous membrane and then degassed by ultrasonication.

[0051] As an optional implementation, the pore size of the microporous filter membrane is 0.20~0.25 μm, for example, it can be 0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, etc.

[0052] As an optional implementation, the power of the ultrasonic degassing is 400~600 W, for example, 400 W, 450 W, 500 W, 550 W, 600 W, etc., the frequency is 40~60 kHz, for example, 40 kHz, 45 kHz, 50 kHz, 55 kHz, 60 kHz, etc., and the ultrasonic degassing time is 5~30 min, for example, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, etc.

[0053] As an optional implementation, the liquid chromatography-mass spectrometry oligonucleotide sequence confirmation method includes the following steps: (1) Preparation of sample solution: Weigh the sample and place it in a volumetric flask, dissolve it, make up to volume, and dilute to obtain the sample solution; (2) Prepare mobile phase A and mobile phase B, filter them through a microporous membrane, degas them by ultrasonication, and then detect the sample solution using the IPRP-LC-MS / MS method; (3) The data is processed using CONFIRM Sequence to obtain the sequence matching rate.

[0054] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0055] Example 1 This embodiment provides a method for confirming oligonucleotide sequences using liquid chromatography-mass spectrometry, the method comprising: 1. Instruments and raw materials used (1) Waters ACQUITY Premier UPLC, equipped with an ultraviolet detector and full-wavelength detection; (2) Waters Xevo G3 QTof mass spectrometry system; (3) Chromatographic column: Waters Acquity Premier OST BEH C18 column, 3.5 μm, 2.1 × 100 mm; (4) Ultrasonic degassing device; (5) The ASO nucleic acid sample was obtained from Notenol and consisted of 18 nucleotides linked by a phosphate thioester backbone (its structural formula is shown in Figure 1). Figure 2 (As shown).

[0056] 2. Testing conditions (1) Diluent / Blank: Water; (2) Detection wavelength: 260 nm; (3) Flow rate: 0.4 mL / min; (4) Injection volume: 10 µL; (5) Column temperature: 40 ℃; (6) Sample tray temperature: 5 ℃; (7) Mobile phase A: 10 mM dibutylamine acetate, pH 8.0, 10% acetonitrile; (8) Mobile phase B: 10 mM dibutylamine acetate, pH 8.0, 80% acetonitrile; (9) The elution gradient is shown in Table 1 below: Table 1

[0057] (10) Ionization mode: ESI; (11) Scan mode: Negative polarity; (12) Analyzer mode: Sensitivity mode; (13) Precursor ion: Precursor m / z: 1782.06; (14) Capillary voltage: 1.5 kV; (15) Sample cone voltage: 40 V; (16) Cone gas flow rate: 50 L / h; (17) Desolvation gas flow rate: 800 L / h; (18) Ion source temperature: 100 °C; (19) Desolvation temperature: 350 °C; (20) Mass range m / z: 500-2000; (21) Collision energy mode: 35 V (22) Scan time: 1 s.

[0058] 3. Detection method: (1) Weigh approximately 20 mg of the test sample accurately, place it in a 50 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well; (2) Prepare mobile phase A and mobile phase B, filter them through a microporous membrane (0.22 μm), degas them by sonication at a frequency of 40 kHz for 15 min, and then detect the sample solution by IPRP-LC-MS / MS method; (3) After the detection is completed, use “CONFIRM Sequence” to process the data, perform fragment matching based on the sample data and theoretical secondary fragment results, and obtain the sequence coverage.

[0059] Figure 1 The matching results after processing the nucleic acid sample detection data show 100% sequence coverage, and at least one pair of complementary fragment ions can match at each break site. This demonstrates that the analytical method provided by this invention is highly direct, precise, high-resolution, and accurate. The detection process takes only 15 minutes, and the entire analysis is completed within 10 minutes, offering advantages such as ease of operation, high accuracy, and rapid result acquisition. Furthermore, the mobile phase composition and elution gradient settings in this invention are directly related to the effective elution of the sample, and mass spectrometry parameters (especially collision energy voltage) play a crucial role in sequence matching. These parameters can be adjusted for different oligonucleotide samples; the parameters used in this example are preferred.

[0060] Example 2 This embodiment provides a method for confirming oligonucleotide sequences using liquid chromatography-mass spectrometry. The only difference from Embodiment 1 is that the collision energy voltage is 30 V; the other steps are the same as in Embodiment 1.

[0061] Detection results: Sequence matching rate less than 100%.

[0062] Example 3 This embodiment provides a method for confirming oligonucleotide sequences using liquid chromatography-mass spectrometry. The only difference from Embodiment 1 is that the collision energy voltage is 50 V; the other steps are the same as in Embodiment 1.

[0063] Detection results: Sequence matching rate less than 100%.

[0064] Example 4 This embodiment provides a method for confirming oligonucleotide sequences using liquid chromatography-mass spectrometry. The only difference from Example 1 is that the injection volume is 15 µL, while the other steps are the same as in Example 1.

[0065] Detection results: Sequence matching rate less than 100%.

[0066] Example 5 This embodiment provides a method for confirming oligonucleotide sequences using liquid chromatography-mass spectrometry. The only difference from Example 1 is that the precursor ion is 1783.28 m / z, while the other steps are the same as in Example 1.

[0067] Detection results: Sequence matching rate less than 100%.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for confirming oligonucleotide sequences using liquid chromatography-mass spectrometry, characterized in that, The confirmation method includes: The sample solution containing oligonucleotides was detected using the IPRP-UPLC-MS / MS method to obtain fragment ion data; The fragment ion data were processed using CONFIRM Sequence to obtain the sequence matching rate.

2. The method for oligonucleotide sequence confirmation using liquid chromatography-mass spectrometry according to claim 1, characterized in that, The sample solution containing the oligonucleotide drug was prepared by the following steps: The oligonucleotide to be tested is dissolved in water to obtain the sample solution containing the oligonucleotide; Preferably, the concentration of the oligonucleotide-containing sample solution is 0.1~1.0 mg / mL.

3. The method for oligonucleotide sequence confirmation using liquid chromatography-mass spectrometry according to claim 1, characterized in that, The detection was performed using a Waters Premier ultra-high performance liquid chromatograph for liquid chromatography and a Waters Xevo G3 high-performance time-of-flight tandem mass spectrometer for mass spectrometry.

4. The method for oligonucleotide sequence confirmation using liquid chromatography-mass spectrometry according to claim 1, characterized in that, The chromatographic column used for the detection was an OST BHE C18 column; Preferably, the specifications of the chromatographic column used for the detection include: a column length of 50~150 mm, an inner diameter of 1.5~4.6 mm, and a packing particle size of 1.5~5.0 μm; Preferably, the chromatographic column used for the detection is a Waters Acquity Premier OST BEH C18 column with a length of 100 mm, an inner diameter of 2.1 mm, and a packing particle size of 3.5 μm.

5. The method for oligonucleotide sequence confirmation using liquid chromatography-mass spectrometry according to claim 1, characterized in that, The mobile phase A used in the liquid chromatography of the detection includes dibutylamine acetate, acetonitrile, and water, and the mobile phase B includes dibutylamine acetate, acetonitrile, and water. Preferably, in the mobile phase A, the concentration of dibutylamine acetate is 5-15 mM, the volume percentage of acetonitrile is 5-15%, and the balance is water; Preferably, in the mobile phase B, the concentration of dibutylamine acetate is 5-15 mM, the volume percentage of acetonitrile is 75-90%, and the balance is water; Preferably, the pH of mobile phase A and mobile phase B are each independently 7.0 to 8.

0.

6. The method for oligonucleotide sequence confirmation using liquid chromatography-mass spectrometry according to claim 1, characterized in that, The gradient elution conditions used in the liquid chromatography for the detection are as follows: 0 min, mobile phase A: 85~95%, mobile phase B: 5~15%; 2 min, mobile phase A: 85~95%, mobile phase B: 5~15%; 12 min, mobile phase A: 5~15%; mobile phase B: 85~95%; 12.1 min, mobile phase A: 85~95%; mobile phase B: 5~15%; 15 min, mobile phase A: 85~95%; mobile phase B: 5~15%; Preferably, the gradient elution conditions used in the liquid chromatography during the detection are as follows: 0 min, mobile phase A: 90%, mobile phase B: 10%; 2 min, mobile phase A: 90%, mobile phase B: 10%; 12 min, mobile phase A: 10% : mobile phase B: 90%; 12.1 min, mobile phase A: 90%; mobile phase B: 10%; 15 min, mobile phase A: 90% : mobile phase B: 10%.

7. The method for oligonucleotide sequence confirmation using liquid chromatography-mass spectrometry according to claim 1, characterized in that, The conditions used in the liquid chromatography for the detection include: wavelength of 250~270 nm; flow rate of 0.1~1.0 mL / min; injection volume of 3~10 μL; column temperature of 35~70℃; and injection plate temperature of 2~10℃.

8. The method for oligonucleotide sequence confirmation using liquid chromatography-mass spectrometry according to claim 1, characterized in that, The mass spectrometry conditions used in the detection included: ionization mode: ESI; scanning mode: negative polarity; analysis mode: sensitivity mode; precursor ion: 1782.06 m / z; capillary voltage: 1.5~2.5 kV; sample cone voltage: 40~100 V; cone gas flow rate: 45~55 L / h; desolvation gas flow rate: 700~900 L / h; ion source temperature: 100~150℃; desolvation temperature: 300~450℃; mass range: 500~3000 m / z; collision energy voltage: 10~50 V; and scan time: 0.5~2 s.

9. The method for oligonucleotide sequence confirmation using liquid chromatography-mass spectrometry according to claim 1, characterized in that, After preparing the mobile phase A and mobile phase B, the following post-processing steps are required; Mobile phase A and mobile phase B were filtered through a microporous membrane and then degassed by ultrasound.

10. The method for oligonucleotide sequence confirmation using liquid chromatography-mass spectrometry according to claim 9, characterized in that, The microporous filter membrane has a pore size of 0.20~0.25 μm; Preferably, the ultrasonic degassing power is 400~600 W, the frequency is 40~60 kHz, and the ultrasonic degassing time is 5~30 min.

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