Method for analyzing double-stranded oligonucleotides

Abstract

A method for analyzing a double-stranded oligonucleotide using an ion trap-type mass spectrometry device provided with an ion source using a matrix-assisted laser desorption ionization method, an ion trapping unit for trapping and discharging ions generated in the ion source, and a detection unit for detecting the ions discharged from the ion trapping unit, a method for analyzing a double-stranded oligonucleotide contained in a sample by introducing a sample into an ion source and irradiating the sample with laser light, capturing ions generated in the ion source in an ion capturing unit, and then discharging and detecting the ions captured by the ion capturing unit, the delay time, which is the time from the irradiation of laser light to the sample by the ion source to the application of a capture voltage for capturing ions to the ion capture unit, is set to be a delay time. The delay time for capturing ions having a mass-to-charge ratio that is the same as the mass-to-charge ratio corresponding to the molecular weight of the double-stranded oligonucleotide is longer than a value that is set in advance for the ion trap-type mass spectrometric analyzer as the delay time for capturing ions having a mass-to-charge ratio that is the same as the mass-to-charge ratio. As a result, a double-stranded oligonucleotide can be analyzed.

Description

Technical Field

[0001] This invention relates to an analytical method for double-stranded oligonucleotides. Background Technology

[0002] Nucleic acid drugs, with DNA or RNA as their basic backbone, are highly specific because they can bind to specific base sequences on genes and thus are expected to be used to treat cancers or hereditary diseases that are currently difficult to treat. There are various types of nucleic acid drugs, among which, in recent years, many have been developed that consist of double-stranded RNA composed of more than twenty base pairs, namely siRNA (small interfering RNA).

[0003] The double-stranded structure of nucleic acids is stable in the liquid phase through hydrogen bonds between base pairs and stacking interactions caused by base overlap, but it is difficult to maintain this double-stranded structure in the gas phase. On the other hand, in mass spectrometry analysis using electrospray ionization (ESI) or matrix-assisted laser desorption / ionization (MALDI), sample molecules are ionized in the gas phase. Furthermore, in MALDI mass spectrometry analysis, the double strands are easily separated due to the influence of the matrix, solvent, and crystallization process used in the preparation of the analyte. Therefore, detecting double-stranded oligonucleotides such as siRNA using these mass spectrometry analyses is difficult.

[0004] To address the aforementioned issues, Non-Patent Literature 1 describes a method for detecting siRNA by using an analytical sample prepared with a neutral matrix instead of an acidic matrix that readily interacts with hydrogen bonds, and performing MALDI mass spectrometry analysis. Specifically, the analytical sample is prepared using 6-aza-2-thiothymine (ATT: 6-aza-2-thiothymine) as the matrix, ammonium citrate as the matrix additive, and water as the solvent. The siRNA is then analyzed using MALDI-TOFMS, which combines a MALDI ion source with a time-of-flight mass spectrometer (TOFMS).

[0005] Existing technical documents Patent documents Non-patent literature 1: Ute Bahr et al., “Detection and Relative Quantification of siRNA Double Strands by MALDI Mass Spectrometry”, Analytical Chemistry, 2008, Vol. 80, No. 16, pp. 6280-6285. Summary of the Invention The technical problem that the invention aims to solve In recent years, attempts have been made to use MALDI-ITMS, a combination of MALDI ion sources and ion trap mass spectrometers (ITMS), for the analysis of single-stranded oligonucleotides. However, even under conditions suitable for analyzing single-stranded oligonucleotides, double-stranded oligonucleotides cannot be analyzed using MALDI-ITMS.

[0006] The technical problem to be solved by the present invention is to provide a new method for analyzing double-stranded oligonucleotides using a MALDI ion trap mass spectrometer.

[0007] Solution to the above technical problems To solve the above-mentioned technical problems, the analytical method for double-stranded oligonucleotides involved in this invention is as follows: An ion trap mass spectrometry apparatus, comprising an ion source based on matrix-assisted laser desorption / ionization, an ion trapping unit for capturing and removing ions generated in the ion source, and a detection unit for detecting ions removed from the ion trapping unit, is used to analyze double-stranded oligonucleotides contained in the sample. The sample is introduced into the ion source and irradiated with a laser. Ions generated in the ion source are captured by the ion trapping unit, and the captured ions are removed and detected. The feature is that the time from when the sample is irradiated with laser from the ion source to when a capture voltage for capturing ions is applied to the ion capture unit, i.e., the delay time, is set to be longer than a value preset for the ion trap mass spectrometer as the delay time for capturing ions having a mass-to-charge ratio similar to that corresponding to the molecular weight of the double-stranded oligonucleotide.

[0008] Invention Effects According to the present invention, double-stranded oligonucleotides can be analyzed using a MALDI ion trap mass spectrometer. Attached Figure Description

[0009] Figure 1This is a schematic diagram illustrating an example of a mass spectrometry analysis apparatus (MALDI-ITMS) used in the analytical method for double-stranded oligonucleotides according to an embodiment of the present invention.

[0010] Figure 2 This is a mass spectrum of vutrisiran under various set conditions in Example 1 (Experimental Example 1-1).

[0011] Figure 3 This is a mass spectrum of vutrisiran under various other settings in Example 1 (Experimental Example 1-1).

[0012] Figure 4 This is a mass spectrum of vutrisiran in Example 1 (Experimental Examples 1-2).

[0013] Figure 5 This is a mass spectrum of vutrisiran in Example 1 (Experimental Examples 1-3).

[0014] Figure 6 This is a mass spectrum of vutrisiran in Example 2 (Experimental Example 2-1).

[0015] Figure 7 This is a mass spectrum of vutrisiran in Example 2 (Experimental Example 2-2).

[0016] Figure 8 This is a mass spectrum of vutrisiran in Example 2 (Experimental Examples 2-3).

[0017] Figure 9 This is the mass spectrum of patisiran under various set conditions in Example 3.

[0018] Figure 10 This is the mass spectrum of patisiran under various other settings in Example 3.

[0019] Figure 11 It is the mass spectrum of patisiran under various other set conditions in Example 3. Detailed Implementation

[0020] The following describes one embodiment of the analytical method for double-stranded oligonucleotides involved in this invention.

[0021] Mass spectrometry analysis device Figure 1A schematic diagram of the mass spectrometry analysis apparatus used in this embodiment is shown. This mass spectrometry analysis apparatus is a MALDI-ITMS, which combines a MALDI ion source and an ion trap type mass spectrometry analysis apparatus. It includes: an ion source 1 for ionizing a sample containing the analyte; an ion trap 2 for temporarily capturing ions with a predetermined mass-to-charge ratio (m / z) generated in the ion source 1 by means of a high-frequency electric field, and separating the captured ions according to the mass-to-charge ratio; and a detection unit 3 for detecting the separated ions.

[0022] (Ion source) Ion source 1 is an ion source using the MALDI method, including a laser irradiation unit 11 for irradiating the sample with laser light, and a sample stage 12 for placing a sample plate S carrying the sample.

[0023] (Ion trap) The ion trap 2 is a quadrupole ion trap comprising a ring-shaped electrode 21 and a pair of end cap electrodes 22 and 23 disposed opposite to each other across the ring electrode 21. An ion injection hole 22a is formed on the inlet-side end cap electrode 22, and an ion emission hole 23a is formed on the outlet-side end cap electrode 23.

[0024] A predetermined high-frequency voltage is applied to the annular electrode 21 and end cap electrodes 22 and 23 of the ion trap 2. A high-frequency electric field is formed in the ion trap 2 where the high-frequency voltage is applied, thereby allowing ions to be captured in the internal space surrounded by the annular electrode 21 and end cap electrodes 22 and 23, or to be ejected from this internal space through the ion ejection port 23a. In this embodiment, the ITMS utilizes the mass separation function of the ion trap 2 itself to sequentially eject ions captured in the ion trap 2 in ascending order of mass-to-charge ratio, and the ions are detected by a detector 3 located outside the ion trap 2. Alternatively, the ITMS can also be a mass spectrometry device that separates ions simultaneously ejected from the ion trap 2 according to their mass-to-charge ratio using a mass separation unit located outside the ion trap 2, such as a time-of-flight mass separation unit, and detects them using a detector 3 located outside the ion trap 2.

[0025] The high-frequency voltage applied to the annular electrode 21 and the end cap electrodes 22 and 23 can be a sinusoidal voltage or a rectangular wave voltage. An ion trap mass spectrometer that utilizes the electric field formed by the rectangular wave voltage is called a digital ion trap mass spectrometer (DITMS).

[0026] When the high-frequency voltage is a sinusoidal voltage, it is difficult to change the frequency because a resonator is required in the voltage generator that produces the sinusoidal voltage. Therefore, the mass-to-charge ratio range of the ions trapped in ion trap 2 is controlled by changing the amplitude of the voltage.

[0027] On the other hand, when the high-frequency voltage is a rectangular wave voltage, a rectangular wave voltage can be generated by rapidly switching between two different voltages even without using a resonator in the voltage generator. Therefore, in DITMS, the mass-to-charge ratio range of ions trapped in ion trap 2 can be controlled by changing the frequency while keeping the voltage amplitude constant. Specifically, by adjusting the frequency of the high-frequency voltage, the low mass-to-charge ratio cutoff value (LMCO) is set; the higher the frequency, the smaller the LMCO is set. Since the amount of ions that ion trap 2 can trap is limited, the mass-to-charge ratio range of ions trapped in ion trap 2 (i.e., the mass-to-charge ratio range measured by DITMS) is set on the low mass-to-charge ratio side when the frequency increases and the LMCO is set smaller; and on the high mass-to-charge ratio side when the frequency decreases and the LMCO is set larger.

[0028] (Detector) The detector 3 includes a conversion multiplier 31 that converts ions into electrons, and a secondary electron multiplier tube 32 that multiplies the electrons from the conversion multiplier 31. A high voltage with the opposite polarity to the ion to be detected is applied to the conversion multiplier 31; that is, a negative voltage is applied to positive ions, and a positive voltage is applied to negative ions. Furthermore, the secondary electron multiplier tube 32 has multiple or continuously arranged multipliers. By applying a voltage to the secondary electron multiplier tube 32, the secondary electrons released from the incident electrons from the conversion multiplier 31 are repeatedly multiplied. The increased secondary electrons are then converted into a current signal and detected. The voltages applied to the conversion multiplier 31 and the secondary electron multiplier tube 32 are predetermined values ​​inherent to the device, based on the mass-to-charge ratio range of the ions trapped in the ion trap 2.

[0029] Through careful research, the inventors of this application discovered that by appropriately setting the time (hereinafter referred to as the delay time) from the time the ion source 1 irradiates the sample with laser to the time a high-frequency voltage is applied to the ring electrode 21 of the ion trap 2 in MALDI-ITMS, double-stranded oligonucleotides can be detected in mass spectrometry analysis. Specifically, the delay time is set to a value preset in MALDI-ITMS that is longer than the delay time used to capture ions with a mass-to-charge ratio similar to that corresponding to the molecular weight of the analyte, i.e., the double-stranded oligonucleotide.

[0030] Generally, when using MALDI mass spectrometry to analyze substances such as peptides or proteins, it is known that [M+H] is the primary substance detected. + Or [MH] - (M represents a molecule, H represents a hydrogen atom) Ions with similar molecular weights. For example, to detect [M+H] ions of a substance with a molecular weight of approximately 15000. + Or [MH]- Typically, the delay time is set to approximately 25 μs. However, at this delay time, double-stranded oligonucleotides with the same molecular weight cannot be detected. On the other hand, when the delay time is set to, for example, a longer value of 80 μs, peaks originating from double-stranded oligonucleotides are confirmed to be detected. Generally, the longer the delay time, the more preferentially ions on the high mass-to-charge ratio side are captured to ion trap 2, but the aforementioned delay time value (80 μs) preferentially captures [M+H] molecules with a molecular weight much larger than approximately 15,000. + Or [MH] - The value of the mass-to-charge ratio of the ion. That is, the delay time of 80 μs exceeds the range that was generally expected based on the relationship between the mass-to-charge ratio of the detected ion and the delay time.

[0031] The delay time can be set to an appropriate value based on the molecular weight of the double-stranded oligonucleotide. For example, setting the delay time to a value in the range of 40 to 80 μs allows for the high-sensitivity detection of double-stranded oligonucleotides with a molecular weight of 10,000 or higher. From the perspective of improving sensitivity, a value of 60 to 80 μs within the above range is more preferable. Considering that the typical delay time for preferentially capturing ions with a mass-to-charge ratio of approximately 20,000 to 70,000 to ion trap 2 is approximately 50 μs, it can be understood that the above-mentioned delay time range is beyond expectations. In addition, in this specification, the numerical range from the lower limit to the upper limit is expressed by "(lower limit) ~ (upper limit)" and "~", and the numerical range expressed in this way includes the lower limit itself and the upper limit itself.

[0032] In addition to setting the delay time, at least one of the voltage applied to the dynode 31 of detector 3 and the voltage of the secondary electron multiplier tube 32 can be set to a value higher than the inherent predetermined value of the device, thereby increasing the signal intensity of the ions detected by detector 3. This allows for the detection of double-stranded oligonucleotides with higher sensitivity. Hereinafter, the voltage applied to the dynode 31 will be referred to as the dynode voltage, and the voltage applied to the secondary electron multiplier tube 32 will be referred to as the detector voltage. The dynode voltage is preferably set to be about 5 to 30% higher than the inherent predetermined value of the device. Furthermore, the detector voltage is preferably set to be about 5 to 50% higher than the inherent predetermined value of the device. For example, if the predetermined value is 7000V, the dynode voltage can be set to about 8000V; if the predetermined value is 1300V, the detector voltage can be set to about 1600 to 2000V.

[0033] <Analytical Samples> The analytical sample is prepared by mixing a sample solution containing double-stranded oligonucleotides with a matrix solution containing the matrix, and then drying the mixture on the sample plate S of the mass spectrometer. Alternatively, the sample solution and matrix solution can be prepared in advance, added dropwise to the sample plate S, and dried; or the sample solution and matrix solution can be added dropwise to the sample plate S separately, mixed on the sample plate S, and then dried.

[0034] (Double-stranded oligonucleotides) The double-stranded oligonucleotide used as the analytical target in this embodiment can be either DNA or RNA. There is no particular limitation on the length of the double-stranded oligonucleotide, which may be, for example, around several tens of base pairs. The double-stranded oligonucleotide can be a chemically synthesized artificial nucleic acid, such as a nucleic acid drug, or a natural substance or its processed product obtained from an organism.

[0035] The molecular weight of the double-stranded oligonucleotide being analyzed can be unknown or known. When the structure of the double-stranded oligonucleotide is known, the molecular weight can be calculated from its chemical formula. Alternatively, the molecular weight can be determined in advance using non-mass spectrometry methods such as electrophoresis. For example, when the double-stranded oligonucleotide is siRNA, its molecular weight is approximately 1000–30000.

[0036] (Matrix) As a matrix, appropriate substances can be selected according to the type of double-stranded oligonucleotide. For example, 3-hydroxypicolinic acid (3-HPA), 2,4-dihydroxyacetophenone (2,4-DHAP), 2,5-dihydroxybenzoic acid (DHB), 2',4',6'-trihydroxyacetophenonemonohydrate (THAP), 6-aza-2-thiothymine (ATT), 3-aminopyrazine-2-carboxylic acid (APCA), anthranilic acid (AA), and nicotinic acid (NA) can be used. From the perspective of being able to detect double-stranded nucleotides with high sensitivity, 3-HPA, DHAP and THAP are preferred as matrices, with 3-HPA being particularly preferred.

[0037] The matrix solution may also be a mixed matrix solution containing two or more matrices. The mixed matrix solution preferably contains at least 3-HPA, more preferably contains 3-HPA and THAP or 3-HPA and 2,4-DHAP.

[0038] There are no particular restrictions on the concentration of the matrix in the matrix solution, but [M+H] can be detected with high sensitivity in double-stranded oligonucleotides. + Or [MH] - From the perspective of [the specific concentration], the preferred concentration is 30–50 mg / mL.

[0039] (Matrix additives) The matrix solution may further contain a matrix additive. As a matrix additive, diammonium citrate dibasic (ACD) can be used. Ammonium salts of citric acid come in several types depending on the number of ammonium ions bound to the citrate ion, but in this embodiment, a salt in which one citrate ion binds to two ammonium ions is preferred.

[0040] There are no particular limitations on the concentration of matrix additives in the matrix solution, but [M+H] can be detected with high sensitivity in double-stranded oligonucleotides. + Or [MH] - From the angle of view, it is preferably 70-200mM, more preferably 100-200mM.

[0041] (solvent) Double-stranded oligonucleotides are water-soluble, and their double-stranded structure is stabilized in the liquid phase through hydrogen bonds. Therefore, water is preferably used as the solvent for the sample solution. Furthermore, the solvent for the matrix solution preferably contains water, but polar solvents such as acetonitrile may also be used.

[0042] The following examples illustrate the analytical method for double-stranded oligonucleotides involved in this invention, but these are merely illustrative and the invention is not limited thereto.

[0043] Example 1 (Experimental Example 1-1) <1. Preparation of Sample Solution> As a sample solution, vutrisiran (siRNA (a double-stranded RNA composed of a sense strand and an antisense strand, molecular weight 16344.55 Da, a research synthetic nucleic acid manufactured by MedchemExpress) was prepared (sense strand: 5'-Ums-Gms-Gm-Gm-Am-Um-Uf-Um-Cf-Af-Uf-Gm-Um-Am-Am-Cm-Cm-Am-Am-Gm-Am-R1-3' (Af=2'-fluoroadenosine, Am=2'-O-methyladenosine, Cf=2'-fluorocytidine, Cm=2'-O-methylcytidine, Gm=2'-O-methylguanosine, Uf=2'-fluorouridine, Um=2'-O-methyluridine, R1=3-branched N-acetylgalactose)). A 20 pmol / μL aqueous solution of amine (GalNAc, s- (hyphen) = thiophosphate bond), sequence number 1, molecular weight 8788.55 Da (antense chain: 3'-Cms-Ums-Am-Cm-Cm-Cm-Um-Af-Am-Af-Gm-Um-Am-Cm-Af-Um-Um-Gf-Gm-Um-Ums-Cfs-Um-5' (Af=2'-fluoroadenosine, Am=2'-O-methyladenosine, Cf=2'-fluorocytidine, Cm=2'-O-methylcytidine, Gf=2'-fluoroguanosine, Gm=2'-O-methylguanosine, Um=2'-O-methyluridine, s- (hyphen) = thiophosphate bond), sequence number 2, molecular weight 7556.00 Da).

[0044] <2. Preparation of matrix solution> As a matrix additive, diammonium citrate (ACD) was dissolved in a 50% acetonitrile aqueous solution to achieve a concentration of 200 mM, thus preparing an ACD solution. In this ACD solution, 3-hydroxypyridinecarboxylic acid (3-HPA) was dissolved to a concentration of 40 mg / mL, thus preparing a matrix solution.

[0045] <3. Preparation of Analytical Samples> The sample solution prepared in step 1. is mixed with the matrix solution prepared in step 2. at a ratio of 1:1 (v / v). 1 μL of the resulting mixture is dropped onto the sample plate (SUS plate) and dried.

[0046] <4. Mass Spectrometry Analysis> Mass spectrometry analysis was performed using a MALDI digital ion trap mass spectrometer (MALDI-DITMS, manufactured by Shimadzu Corporation, trade name: MALDImini-1). The sample plate from step 3 was inserted into the MALDI-DITMS, and measurements were performed in positive ion mode using the grating function. Measurement conditions were as follows: based on the molecular weight of the siRNA contained in the analytical sample, a mass-to-charge ratio range of mode 3 (m / z 2000-18000) was used. Measurements were performed using the optimal laser power (threshold) at the device's pre-set parameters (detector voltage (DV1): 1300 (V), dynode voltage (DV2): 7000 (V), RF delay (RF): 25 (μs)). Furthermore, measurements were performed after appropriately adjusting the detector voltage (DV1), dynode voltage (DV2), and delay time (RF).

[0047] <Results> Figure 2 The mass spectra of vutrisiran are shown when 3-HPA is used as the matrix.

[0048] When measurements were performed under the device's inherent settings (DV1: 1300, DV2: 7000, RF: 25), two peaks were primarily detected originating from the sense and antisense strands, both ending in [M+H]. + The form appeared, but no peak originating from the double strand was detected. Figure 2 (a)). On the other hand, when the values ​​of the detector voltage (DV1) and the dynode voltage (DV2) are increased, and measurements are performed under conditions that enhance signal strength (DV1: 1800, DV2: 8000, RF: 25), a peak originating from the double strand with [M+H] is detected. + It appears in various forms, but its sensitivity is low. Figure 2 (b)). Further measurements were performed under conditions of increased delay time (RF) values ​​(DV1: 1800, DV2: 8000, RF: 80), enabling high-sensitivity detection of peaks originating from the double strand. Figure 2 (c)).

[0049] Figure 3The mass spectra of vutrisiran are shown with 3-HPA as the matrix, detector voltage set to DV1: 1800, dynode voltage set to DV2: 8000, and the delay time (RF) value varied. The mass spectra from bottom to top are for RF: 25, 40, 60, 80, and 100, with magnified views of the m / z values ​​of the peaks originating from the double strands displayed on the right side of each layer. Arrows in the figure indicate the detection of peaks originating from the double strands, and the values ​​in the magnified views represent the peak intensity (mV).

[0050] from Figure 3 It can be seen that regardless of the delay time (RF) value, peaks originating from the double strand are detected, but the noise is relatively large and the peak intensity is low at RF values ​​of 25 and 100. Figure 3 (a) and (e)). On the other hand, the noise is lower at RF: 40–80 ( Figure 3 (b) to (d)), especially at RF: 60 and 80, peaks originating from the double strand can be detected with high sensitivity. Figure 3 (c) and (d)).

[0051] (Experimental Examples 1-2) Except for the different preparation method of the matrix solution, mass spectrometry analysis was performed using the same method as in Experiment 1-1.

[0052] <2. Preparation of matrix solution> As a matrix additive, ACD was dissolved in a 50% acetonitrile aqueous solution to a concentration of 200 mM to prepare an ACD solution. In this ACD solution, 3-HPA, 2,4-dihydroxyacetophenone (DHAP), or 2',4',6'-trihydroxyacetophenone monohydrate (THAP) were dissolved as matrices to a concentration of 40 mg / mL, respectively, to prepare three matrix solutions (3-HPA solution, DHAP solution, and THAP solution).

[0053] Two mixed matrix solutions (3-HPA / THAP (1:1) solution and 3-HPA / THAP (1:3) solution) were prepared by mixing 3-HPA solution with THAP solution at a ratio of 1:1 (v / v) and 1:3 (v / v).

[0054] <Results> Figure 4The mass spectra of vutrisiran are shown using various matrices (DHAP, THAP, 3-HPA / THAP (1:1), 3-HPA / THAP (1:3)). The mass spectra on the left were measured using the device's inherent settings (DV1: 1300, DV2: 7000, RF: 25), while the mass spectra on the right were measured using the conditions in Experimental Example 1-1 where the peaks originating from the double strand were detected with high sensitivity (DV1: 1800, DV2: 8000, RF: 80) (hereinafter referred to as the double strand detection settings). The arrows in the figure indicate the detection status of peaks originating from the double strand, and ND indicates that they were not detected. For peaks originating from the double strand, a magnified view of the m / z region of the peak is further shown.

[0055] Similar to when 3-HPA was used as the matrix, peaks originating from double strands were detected regardless of the matrix used, provided that the detector voltage (DV1), dynode voltage (DV2), and delay time (RF) were set to values ​​greater than the inherent settings of the device. Figures 2-4 It can be confirmed that siRNA detection sensitivity is highest when 3-HPA is used as the matrix.

[0056] (Experimental Examples 1-3) Except for the different preparation method of the matrix solution, mass spectrometry analysis was performed using the same method as in Experiment 1-1 of Example 1.

[0057] <2. Preparation of matrix solution> As a matrix additive, ACD was dissolved in 50% acetonitrile aqueous solution to prepare five ACD solutions with concentrations of 70, 100, 200, 300, and 400 mM. In these five ACD solutions, 3-HPA was dissolved as a matrix to a concentration of 40 mg / mL to prepare five matrix solutions.

[0058] <Results> Figure 5 The mass spectra of vutrisiran are shown with 3-HPA as the matrix and varying ACD concentrations in the matrix solution. From bottom to top, the mass spectra are for ACD concentrations of 70, 100, 200, 300, and 400 mM. The left side shows the mass spectra obtained using the device's default settings, and the right side shows the mass spectra obtained using double-strand detection settings. The right-hand mass spectra further show magnified views of the m / z region of the peak originating from the double strand, and for peaks originating from the double strand, the peak intensity (mV) is shown.

[0059] from Figure 5It was found that peaks originating from double strands were detected using the double-strand detection settings when the ACD concentration was between 70 and 200 mM. Furthermore, the peak intensity of the double-stranded peaks was higher when using a matrix solution of 200 mM ACD compared to when using a matrix solution of 70 mM ACD.

[0060] Detailed data are not shown here, but in the analysis of single-stranded oligonucleotides, ACD 70mM is the optimal concentration. It has been reported that double-stranded oligonucleotides contain less sodium ions (Na+) in the sample compared to single-stranded oligonucleotides. + More, Na + High concentrations can inhibit sample ionization. In this experimental example, when the ACD concentration was 200 mM, the peak intensity of the double-stranded peak was higher than that at 70 mM. One possible reason is that it is believed to have the ability to capture Na+. + The higher the concentration of ACD that is active, the more difficult it is to prevent ionization.

[0061] In summary, setting the detector voltage (DV1), dynode voltage (DV2), and delay time (RF) values ​​higher than the device's inherent settings is effective for detecting double-stranded oligonucleotides. Under these double-strand detection settings, peaks originating from the double strand can be detected using various matrices. 3-HPA is the most preferred matrix. Furthermore, it has been confirmed that detecting peaks originating from the double strand with higher sensitivity occurs when the concentration of diammonium citrate, used as a matrix additive, is higher than the optimal concentration for single-stranded oligonucleotide analysis.

[0062] Example 2 (Experimental Example 2-1) Except for the different preparation method of the matrix solution, mass spectrometry analysis was performed using the same method as in Experiment 1-1 of Example 1.

[0063] <2. Preparation of matrix solution> As a matrix additive, ACD was dissolved in a 50% acetonitrile aqueous solution to a concentration of 200 mM to prepare an ACD solution. In this ACD solution, 3-HPA or 6-aza-2-thiothymidine (ATT) was dissolved as a matrix to a concentration of 40 mg / mL to prepare two matrix solutions (3-HPA solution and ATT solution).

[0064] <Results> Figure 6 The mass spectra of vutrisiran are shown when 3-HPA or ATT is used as the matrix. The upper layer is the mass spectrum using 3-HPA, the lower layer is the mass spectrum using ATT, the left side is the mass spectrum obtained using the device's inherent settings, and the right side is the mass spectrum obtained using the double-strand detection settings. The R value in the figure indicates the resolution of the peaks originating from the double strand.

[0065] Non-patent literature 1 describes that double-strand peaks are more easily detected using a neutral matrix ATT compared to an acidic matrix 3-HPA. However, in this experimental example, although peaks originating from double strands were detected under both matrices using the double-strand detection settings, the mass spectrometry using ATT had more noise and lower peak resolution compared to the mass spectrometry using 3-HPA, resulting in poorer overall mass spectrometry quality.

[0066] (Experimental Example 2-2) Except for the different preparation method of the matrix solution, mass spectrometry analysis was performed using the same method as in Experiment 1-1 of Example 1.

[0067] <2. Preparation of matrix solution> As a matrix additive, ACD was dissolved in 50% acetonitrile aqueous solution or water to achieve a concentration of 200 mM, thus preparing two ACD solutions. 3-HPA was then dissolved in these two ACD solutions as a matrix to achieve a concentration of 40 mg / mL, thus preparing two matrix solutions (3-HPA solution and 3-HPA aqueous solution).

[0068] <Results> Figure 7 The mass spectra of vutrisiran are shown when 3-HPA is used as the matrix and 50% aqueous acetonitrile solution or water is used as the solvent for the matrix solution. The upper layer is the mass spectrum using 50% aqueous acetonitrile solution, and the lower layer is the mass spectrum using water. The left side shows the results measured using the device's inherent set conditions, and the right side shows the results measured using the set conditions for double-strand detection.

[0069] According to Non-Patent Literature 1, acetonitrile can improve the solubility of the matrix, but it inhibits the detection of double-stranded oligonucleotides. However, in this experimental example, regardless of whether a 50% aqueous solution of acetonitrile or water was used as the solvent, under the conditions set for double-strand detection, it was confirmed that peaks originating from the double strand could be detected with good sensitivity.

[0070] (Experimental Example 2-3) Except for the different preparation method of the matrix solution, mass spectrometry analysis was performed using the same method as in Experiment 1-1 of Example 1.

[0071] <2. Preparation of matrix solution> As a matrix additive, ACD was dissolved in a 50% acetonitrile aqueous solution to achieve a concentration of 200 mM, thus preparing an ACD solution. 3-HPA was then dissolved in this ACD solution as a matrix to achieve a concentration of 40 mg / mL, thus preparing a matrix solution (3-HPA solution).

[0072] In addition, ACD was dissolved in water to achieve concentrations of 100 mM and 200 mM, respectively, to prepare two ACD aqueous solutions. 3-HPA was then dissolved in these two ACD aqueous solutions to achieve concentrations of 40 mg / mL, respectively, to prepare two matrix solutions (3-HPA aqueous solutions).

[0073] As a matrix additive, ACD was dissolved in a 50% acetonitrile aqueous solution to achieve a concentration of 200 mM, thus preparing an ACD solution. ATT was then dissolved in this ACD solution as a matrix to achieve a concentration of 40 mg / mL, thus preparing a matrix solution (ATT solution).

[0074] In addition, ACD was dissolved in water to a concentration of 100 mM to prepare an ACD aqueous solution. ATT was then dissolved in this ACD aqueous solution to a concentration of 6 mg / mL to prepare a matrix solution (ATT aqueous solution).

[0075] <Results> Figure 8 The mass spectra of vutrisiran are shown using various matrix solutions containing 3-HPA or ATT as the matrix. The left side shows the results measured using the instrument's inherent settings, while the right side shows the results measured using the double-strand detection settings. The mass spectrum on the right further shows magnified views of the m / z region of the peak originating from the double strand; the values ​​in the magnified views represent the peak intensity (mV) and resolution (R) of that peak.

[0076] When ATT is used as a matrix ( Figure 8 Peaks originating from the double strand were detected in (d) and (e), but there was significant noise and low resolution. Furthermore, in Figure 8 In (d) and (e), peaks originating from dimers of the sense and antisense strands were detected (in the magnified image, the peak originating from the sense strand dimer is labeled 2ss, and the peak originating from the antisense strand dimer is labeled 2as), as well as multiple peaks originating from base-depleted ions. Additionally, Figure 8 The matrix solution conditions for (e) were the same as those for siRNA detection by MALDI-TOFMS in Non-Patent Literature 1. In contrast, when using 3-HPA ( Figure 8 (a) to (c), especially Figure 8 In (a) and (c)), the double-chain peaks were detected with high sensitivity and high resolution. Furthermore, in Figure 8 In (a) to (c), no peaks originating from dimers were detected, and there were also few peaks originating from base depletion ions, making the mass spectrometry relatively simple.

[0077] In summary, when MALDI-DITMS is set to the conditions for double-strand detection, it was confirmed that using the acidic matrix 3-HPA yields superior mass spectrometry compared to the neutral matrix ATT recommended in Non-Patent Literature 1.

[0078] Example 3 <1. Preparation of Sample Solution> As a sample solution, a 20 pmol / μL aqueous solution of patisiran (siRNA (double-stranded RNA composed of a sense strand and an antisense strand), molecular weight 13424 Da, synthetic nucleic acid for research use manufactured by Gene Design Co., Ltd.) (sense strand: 5'-G-Um-AA-Cm-Cm-AAGAG-Um-A-Um-Um-Cm-Cm-A-Um-dT-dT-3' (dT=thymidine deoxynucleotide, Cm=2'-O-methylcytidine, Um=2'-O-methyluridine), sequence number 3, molecular weight 6764 Da) (antisense strand: 3'-dT-dT-CA-Um-UGGUUCUCA-Um-AAGGUA-5' (dT=thymidine deoxynucleotide, Cm=2'-O-methylcytidine, Um=2'-O-methyluridine), sequence number 4, molecular weight 6660 Da) was prepared.

[0079] <2. Preparation of matrix solution> As a matrix additive, ACD was dissolved in a 50% acetonitrile aqueous solution to achieve a concentration of 70 mM or 200 mM, preparing two ACD solutions. 3-HPA was then dissolved in these two ACD solutions as a matrix to achieve a concentration of 40 mg / mL, preparing two matrix solutions.

[0080] <3. Preparation of Analytical Samples> The sample solution prepared in step 1. was mixed with the matrix solution prepared in step 2. at a ratio of 1:1 (v / v). 1 μL of the resulting mixture was dropped onto the sample plate (SUS plate) and dried.

[0081] <4. Mass Spectrometry Analysis> Mass spectrometry analysis was performed using a MALDI digital ion trap mass spectrometer (MALDI-DITMS, manufactured by Shimadzu Corporation, trade name: MALDImini-1). The sample plate from step 3 was inserted into the MALDI-DITMS, and measurements were performed in positive ion mode using the grating function. Measurement conditions were as follows: the mass-to-charge ratio range corresponding to the molecular weight of the siRNA contained in the analytical sample was used, i.e., mode 3 (m / z 2000-18000). The device's specified settings were: detector voltage (DV1): 1300 (V), dynode voltage (DV2): 7000 (V), and delay time (RF): 25 (μs), with the optimal laser power (threshold) used for measurement. Furthermore, measurements were performed by appropriately varying the detector voltage (DV1), dynode voltage (DV2), and delay time (RF).

[0082] <Results> Figure 9 The mass spectra of patisiran under various set conditions are shown, with 3-HPA used as the matrix and the ACD concentration in the matrix solution varied. The upper layer is the mass spectrum at ACD 70 mM, and the lower layer is the mass spectrum at ACD 200 mM.

[0083] Whether ACD is measured at 70mM or 200mM, under the inherent settings of the device (DV1: 1300, DV2: 7000, RF: 25), two peaks mainly originating from the sense and antisense strands are detected, both ending in [M+H]. + The form appeared, but no peak originating from the double strand was detected. Figure 9 (a)). On the other hand, when measured under conditions of increased delay time (RF) (DV1: 1300, DV2: 7000, RF: 80), peaks originating from the double strand were detected at both ACD 70mM and 200mM, with the [M+H] value. + It appears in the form of [symbol], but the signal-to-noise ratio (S / N) is low. Figure 9 (b)). Under conditions where the detector voltage (DV1) and dynode voltage (DV2) were further increased (DV1: 1800, DV2: 8000, RF: 80), peaks originating from the double strand were detected with good sensitivity at both ACD 70mM and 200mM. Figure 9 (c)). In Figure 9 In (c), the sensitivity of ACD at 200mM is higher than that at 70mM. In summary, by adjusting not only the delay time but also adjusting the detector voltage and dynode voltage to values ​​greater than the inherent settings of the device, peaks originating from double strands can be detected more sensitively.

[0084] Figure 10The mass spectra of patisiran are shown when 3-HPA is used as the matrix, and the detector voltage (DV1) and dynode voltage (DV2) are kept constant at the inherent settings of the device (DV1: 1300, DV2: 7000), with only the delay time (RF) changed. From bottom to top, the RF values ​​are 25, 40, 60, and 80. The left side shows the mass spectra under ACD 70mM conditions, and the right side shows the mass spectra under ACD 200mM conditions.

[0085] from Figure 10 It can be confirmed that, without changing the detector voltage and the dynode voltage, as the delay time increases, peaks originating from the double chain can be detected, but the larger the delay time, the lower the signal-to-noise ratio (S / N).

[0086] on the other hand, Figure 11 The mass spectra of patisiran are shown when the detector voltage (DV1) and dynode voltage (DV2) are increased from the device's inherent settings (DV1: 1800, DV2: 8000), and the delay time (RF) is changed. From bottom to top, the RF values ​​are 25, 40, 60, and 80. The left side shows the mass spectrum at ACD 70 mM, and the right side shows the mass spectrum at ACD 200 mM.

[0087] from Figure 11 It was found that increasing the detector voltage and dynode voltage did not significantly decrease the signal-to-noise ratio (S / N) even when increasing the delay time. Results showed that the highest sensitivity for detecting peaks originating from double strands was achieved at RF: 80, and at this RF value, the sensitivity at ACD 200mM was higher than that at ACD 70mM. Furthermore, it was confirmed that adjusting not only the delay time but also setting the detector voltage and dynode voltage to values ​​greater than the device's inherent settings enabled the detection of peaks originating from double strands with even higher sensitivity.

[0088] [plan] The above exemplary embodiments are specific examples of the following solutions, which will be obvious to those skilled in the art.

[0089] (Item 1) One aspect of the present invention relates to an analytical method for double-stranded oligonucleotides, which is as follows: An ion trap mass spectrometry apparatus, comprising an ion source based on matrix-assisted laser desorption / ionization, an ion trapping unit for capturing and removing ions generated in the ion source, and a detection unit for detecting ions removed from the ion trapping unit, is used to analyze double-stranded oligonucleotides contained in the sample. The sample is introduced into the ion source and irradiated with a laser. Ions generated in the ion source are captured by the ion trapping unit, and the captured ions are removed and detected. The feature is that the time from when the sample is irradiated with laser from the ion source to when a capture voltage for capturing ions is applied to the ion capture unit, i.e., the delay time, is set to be longer than a value preset for the ion trap mass spectrometer as the delay time for capturing ions having a mass-to-charge ratio similar to that corresponding to the molecular weight of the double-stranded oligonucleotide.

[0090] Typically, when using ion trap mass spectrometry, the relationship between the mass-to-charge ratio of the desired ion and the delay time is empirically known. A "preset value" refers to a value based on this empirically known relationship between the mass-to-charge ratio of the desired ion and the delay time, used to capture [M+H] ions with a molecular weight corresponding to the double-stranded oligonucleotide. + Or [MH] - The delay time of ions with the same mass-to-charge ratio is a value preset for the mass spectrometer.

[0091] Furthermore, the molecular weight of the double-stranded oligonucleotide being analyzed can be either unknown or known. When the molecular weight of the double-stranded oligonucleotide being analyzed is known, mass spectrometry analysis is performed within a mass range containing the mass-to-charge ratio corresponding to that molecular weight, with a delay time longer than a pre-set value. The presence and molecular weight information of the double-stranded oligonucleotide can be confirmed by detecting whether a peak providing molecular weight information is detected. On the other hand, when the molecular weight of the double-stranded oligonucleotide being analyzed is unknown, mass spectrometry analysis is performed within multiple candidate mass ranges containing the mass-to-charge ratio corresponding to the expected molecular weight of the double-stranded oligonucleotide, with a delay time longer than a pre-set value. The presence and molecular weight information of the double-stranded oligonucleotide can be confirmed by detecting whether a peak providing molecular weight information is detected.

[0092] According to the analytical method for double-stranded oligonucleotides mentioned in item 1, double-stranded oligonucleotides can be analyzed using a MALDI ion trap mass spectrometer.

[0093] (Item 2) In the analytical method for double-stranded oligonucleotides mentioned in Item 1, it can be set as follows: The detection unit includes a conversion multiplier electrode and a secondary electron multiplier tube. At least one of the voltage applied to the conversion multiplier electrode and the voltage applied to the secondary electron multiplier tube is set to a value higher than a device-specific setting value that is preset according to the mass-to-charge ratio range of ions captured by the ion trapping unit of the ion trapping mass spectrometer.

[0094] According to the analytical method for double-stranded oligonucleotides involved in item 2, double-stranded oligonucleotides can be analyzed with higher sensitivity.

[0095] (Item 3) In the analytical method for double-stranded oligonucleotides involved in Item 1 or Item 2, it can be set as follows: The delay time is 60–80 μs.

[0096] According to the analytical method for double-stranded oligonucleotides involved in item 3, double-stranded oligonucleotides can be analyzed with high sensitivity.

[0097] (Item 4) In any of the analytical methods for double-stranded oligonucleotides involved in items 1 through 3, it is possible to set the method as follows: The ion trap mass spectrometry device is a digital ion trap mass spectrometry device.

[0098] According to the analytical method for double-stranded oligonucleotides described in item 4, double-stranded oligonucleotides can be analyzed with higher sensitivity.

[0099] (Item 5) In any of the analytical methods for double-stranded oligonucleotides involved in items 1 through 4, it is possible to set the method as follows: An analytical sample comprising the sample, a matrix, and diammonium hydrogen citrate as a matrix additive is prepared, and the analytical sample is used for mass spectrometry analysis using the ion trap mass spectrometry analyzer.

[0100] According to the analytical method for double-stranded oligonucleotides described in item 5, it is possible to prepare analytical samples suitable for mass spectrometry analysis using a MALDI ion trap mass spectrometer and to analyze double-stranded oligonucleotides.

[0101] (Item 6) In the analytical method for double-stranded oligonucleotides mentioned in Item 5, it can be set as follows: When preparing a matrix solution comprising the matrix, the diammonium hydrogen citrate, and a solvent, the concentration of the diammonium hydrogen citrate in the matrix solution is 100–200 mM.

[0102] (Item 7) In the analytical method for double-stranded oligonucleotides covered in Item 5 or 6, it can be set as follows: The matrix contains at least 3-hydroxypyridinecarboxylic acid, or 2',4',6'-trihydroxyacetophenone monohydrate or 2,4-dihydroxyacetophenone.

[0103] (Item 8) In any of the analytical methods for double-stranded oligonucleotides involved in items 5 through 7, it is possible to set the method as follows: The solvent is water or a mixture of water and acetonitrile.

[0104] According to the analytical method for double-stranded oligonucleotides involved in any of items 6 to 8, double-stranded oligonucleotides can be analyzed with higher sensitivity.

[0105] (Item 9) In any of the analytical methods for double-stranded oligonucleotides involved in items 1 through 8, it is possible to set the method as follows: The double-stranded oligonucleotide is siRNA.

[0106] Explanation of reference numerals in the attached figures 1…ion source 2…Ion trap 3… Testing Department 11…Laser Irradiation Section 12…Sample Stage 21… Ring electrode 22, 23… End cap electrodes 31…Conversion Multiplier 32…Secondary electron multiplier tube.

Claims

1. A method of analyzing a double-stranded oligonucleotide by using an ion trap type mass spectrometer having an ion source based on a matrix-assisted laser desorption ionization method, an ion trapping section for trapping and discharging ions generated in the ion source, and a detection section for detecting ions discharged from the ion trapping section, introducing a sample into the ion source and irradiating the sample with laser light, trapping ions generated in the ion source into the ion trapping section, discharging and detecting the ions trapped by the ion trapping section, and thereby analyzing a double-stranded oligonucleotide contained in the sample, characterized in that a delay time from the irradiation of the sample with laser light in the ion source until a trapping voltage for trapping ions is applied to the ion trapping section is set to be longer than a value set in advance for the ion trap type mass spectrometer as a delay time for trapping ions having a mass-to-charge ratio in the same order as a mass-to-charge ratio of the double-stranded oligonucleotide.

2. The method of analyzing a double-stranded oligonucleotide according to claim 1, characterized in that the detection section has a conversion dynode and a secondary electron multiplier, and at least one of a voltage applied to the conversion dynode and a voltage applied to the secondary electron multiplier is set to be higher than a value set in advance for the ion trap type mass spectrometer as a value inherent to the device depending on a range of mass-to-charge ratios of ions trapped by the ion trapping section.

3. The method of analyzing a double-stranded oligonucleotide according to claim 1, characterized in that the delay time is 60 to 80 μsec.

4. The method of analyzing a double-stranded oligonucleotide according to claim 1, characterized in that the ion trap type mass spectrometer is a digital ion trap type mass spectrometer.

5. The method of analyzing a double-stranded oligonucleotide according to claim 1, characterized in that an analysis sample containing the sample, a matrix, and diammonium hydrogen citrate as a matrix additive is prepared, and the analysis sample is used for mass spectrometry by the ion trap type mass spectrometer.

6. The method of analyzing a double-stranded oligonucleotide according to claim 5, characterized in that when a matrix solution containing the matrix, the diammonium hydrogen citrate, and a solvent is prepared, the concentration of the diammonium hydrogen citrate in the matrix solution is set to be 100 to 200 mM.

7. The method of analyzing a double-stranded oligonucleotide according to claim 5, characterized in that the matrix contains at least 3-hydroxypyridinecarboxylic acid, or is 2', 4', 6'-trihydroxyacetophenone monohydrate or 2, 4-dihydroxyacetophenone.

8. The method of analyzing a double-stranded oligonucleotide according to claim 5, characterized in that the solvent is water or a mixed solvent of water and acetonitrile.

9. The method of analyzing a double-stranded oligonucleotide according to claim 1, characterized in that the double-stranded oligonucleotide is siRNA. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

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