Negative polarity tuning standard for mass spectrometry

Abstract

The method and composition are used to evaluate the performance of mass spectrometer instruments. [Solution] Oligonucleotides are introduced into a mass spectrometer, the sample is analyzed in negative ionization mode, and the resulting mass spectrometry peaks are compared to known standards of nucleotide-based samples for the purpose of qualifying the negative ionization mode mass spectrometer. The composition is supplied for this purpose and system.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 511,843, filed on 3 July 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] This disclosure relates to a method and composition for assessing the linear response and mass axis calibration of a mass spectrometer in a negative ionization mode through the use of oligonucleotides and novel compositions relating thereto. [Background technology]

[0003] Mass spectrometry (MS) is an important analytical technique used in many scientific fields. It measures the mass-to-charge ratio (m / z) of one or more molecules present in a sample after the ionization process. The measurement results are typically presented as a spectrum, plotting the mass-to-charge (m / z) ratio against the relative abundance of the molecule in question.

[0004] The MS apparatus comprises a detector configured to analyze a sample by measuring the m / z ratio of ions received by the detector. The mass spectrometry results may be presented as a mass spectrum, a plot of intensity corresponding to the mass-to-charge ratio for molecular abundance, or a chromatographic representation of the total amount of observed ions (i.e., a total ion chromatogram (TIC)). Mass spectrometry is useful for identifying and quantifying components of complex mixtures.

[0005] Mass spectrometry analysis generally begins with the ionization of a sample by any number of means, including but not limited to matrix-assisted laser desorption / ionization (MALDI) or electrospray ionization (ES). In MALDI techniques, the sample is embedded in a solid IR or UV-absorbing matrix. The matrix-embedded sample is placed in the ion source of a mass spectrometer. The matrix is ​​vaporized by a short laser pulse, thereby transferring the sample molecules into a gas phase in a non-fragmented state. The sample is ionized by collision and reaction with the matrix ions that are simultaneously generated. A voltage is applied, thereby accelerating the ions into a field-free flight tube. Due to their different masses, the ions in the ion source are accelerated to different speeds, with smaller ions reaching the detector earlier than larger ions. The different flight times translate into different ion masses.

[0006] Electrospray ionization enables the ionization / vaporization of polar molecules. The sample is dissolved in a solvent and then pumped through a narrow capillary tube elevated to a high potential. The low-charged droplets are sprayed at atmospheric pressure from the ES capillary into a buffer gas, descending the pressure and potential gradient towards the opening of the mass spectrometer's high-vacuum system. As the droplets traverse this path, they break down into smaller droplets until ions are removed from the droplets or the solvent is removed.

[0007] Ionization can also generate undesirable adducts from the sample, complicating the quality and resolution of the MS spectrum.

[0008] Ionization may generate positive ions (cations) and negative ions (anions), which can then be introduced into the MS instrument. The mass spectrometer is often used with cationic samples, which is generally referred to as the "positive mode." Alternatively, the mass spectrometer may be used with anionic samples, which is referred to in this disclosure as the "negative ionization mode" or "negative mode."

[0009] To ensure accuracy and reliability, MS instruments need to be qualified. Qualification is a predefined process that determines whether the MS instrument can produce reproducible relative abundance results that follow a linear standard when the concentration or volume of the sample increases. Qualification is a formal demonstration of accuracy, linearity, and other system attributes, which is performed through instrument calibration using established standards as intended by the seller. Generally, an Installation Qualification (IQ) is required for new or used instruments, which is usually based on factory specifications and follows the manufacturer's guidelines. In the Operational Qualification (OQ), after installation, following repeated use, or after major maintenance intervention, the ability of the MS instrument to meet the specified operating criteria is demonstrated and recorded. Performance Qualification (PQ) is the performance specification (i.e., (U)HPLC / MS) defined by the customer or the instrument supplier operating on the system configuration representing its normal operating state. PQ is usually performed after some maintenance intervention.

[0010] Linearity for an MS instrument refers to whether there is a linear relationship between the amount of the sample and the MS signal resulting from that sample. Linearity can be demonstrated by introducing standards at multiple concentrations or volumes and evaluating the MS signals generated by the standards against the known or expected signals from those standards. A perfectly linear result is not necessarily a prerequisite for successful qualification. In some cases, the degree of linearity for successful qualification is defined by the intended use of the MS instrument. Non-linear regression analysis of the results (such as binomial, geometric series, polynomial, logarithmic, geometric series, etc.) may also be used if the sample concentration against the instrument response can be consistently evaluated.

[0011] In addition, the MS instruments must have their mass axes calibrated regularly to ensure the accuracy and reliability of the m / z plots or spectra. Mass axis calibration is a predefined process for determining whether a given mass-to-charge compound is appropriately assigned to the corresponding peak in the MS spectrum. This process consists of analyzing standards on the MS instrument with one or more molecules of known mass and ionization state, and ensuring that the mass peaks match the m / z of the known or expected m / z for that standard. If the resulting mass spectrum matches the known standard, no further maintenance is required. However, if the resulting mass spectrum does not align with the calibration curve or the expected m / z values are not within acceptable tolerances, further maintenance may be required. Mass axis calibration may be performed before analysis, or the mass axis calibration may be adjusted dynamically based on changes observed in the m / z of the standards present throughout the analysis.

[0012] Previous approaches to qualifying negative mode MS instruments have been adversely affected by compounds used in the analysis of oligonucleotides, including 1,1,1,3,3,3-hexafluoro-2-propanol ("HFIP"), triethylamine ("TEA"), hexylamine, dibutylamine, and dihexylamine. Residual ions remaining in the optics and flow paths inside the MS instrument saturate the detector, making currently commercially available standards unusable for assessing accuracy and linearity.

Prior Art Documents

Patent Documents

[0013]

Patent Document 1

Patent Document 2

Patent Document 3

[0014] There is a need for improved methods and standards for qualifying the negative mode of an MS instrument by assessing system response calibration (especially those used to analyze oligonucleotides with ion pair reagents). [Means for Solving the Problems]

[0015] The present disclosure provides novel methods and standards for evaluating the performance of an MS instrument or system. The standards used for evaluation are Formula I, Formula II, Formula III or Formula IV X 1 3´-PL-3´X 2 (I) X 1 5´-PL-5´X 2 (II) X 1 3´-PL-5´X 2 (III) X 1 5´-PL-3´X 2 (IV) comprising one or more oligonucleotides having the structure: wherein PL is a phosphate bond comprising -PO4-, -PO3-O-PO3-, -PO3S, -PO3BH2- or PO3-O-PO2-O-PO3-, X 1 and X 2The standard is independently a nucleoside, nucleotide, or oligonucleotide. In some embodiments, one or more (or all) of the standard oligonucleotides spans a length of 2 to 100 nucleic acid bases. The method or use of the standard may include the step of evaluating one or more mass spectrometry signals from an MS instrument, the evaluation step of including the step of identifying one or more mass peaks corresponding to the standard, and the step of comparing the identified mass peaks with one or more known or expected mass peaks for the standard. The MS instrument has a negative ionization mode, and the standard is introduced as a sample for negative mode mass spectrometry analysis. The method may also include the step of qualifying the mass spectrometer instrument for a negative ionization mode.

[0016] This disclosure also provides novel methods for assessing the mass axis calibration of MS instruments through standardization, or for dynamically adjusting for differences between experimentally measured m / z and theoretical m / z during analysis. [Brief explanation of the drawing]

[0017] [Figure 1] This is a schematic diagram of an example of a mass spectrometry (MS) instrument. [Figure 2] The HPLC chromatogram of the oligonucleotide product synthesized in Example 1 is shown. [Figure 3] The linear curves obtained from MS analysis of oligonucleotides over a certain range of quantities are shown. [Figure 4] Linear curves obtained from MS analysis of oligonucleotides across a different range of quantities are shown. [Figure 5] The linear curve obtained from MS analysis of oligonucleotides containing several fragment ions is shown. [Figure 6] The linear curve obtained from MS analysis of oligonucleotides containing several fragment ions is shown. [Figure 7] The linear curve obtained from MS analysis of oligonucleotides containing several fragment ions is shown. [Modes for carrying out the invention]

[0018] It should be understood that the technical terms used herein are for the purpose of describing only specific embodiments and are not intended to limit them, since the scope of this teaching is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in which this disclosure relates. Defined terms are added to the technical and scientific meanings of defined terms as commonly understood and accepted in the art of this teaching.

[0019] Patents and publications referred to herein are expressly incorporated by reference.

[0020] As used herein, unless otherwise explicitly stated in the context, the terms “one” and “above” include both singular and plural references. For example, “compound” includes both one compound and multiple compounds. “First” and “second” are terms used to distinguish different elements, not to impose numerical limitations, and an apparatus having first and second elements may also include third, fourth, fifth, etc., unless otherwise indicated.

[0021] "Nucleotide" refers to a subunit of an oligonucleotide (whether DNA, RNA, or their analogues) containing a phosphate group, a sugar group, and a heterocyclic base, as well as analogues of such subunits. Other groups (e.g., protecting groups) can also be attached to any component of a nucleotide. "Nucleoside" or "nucleoside moiety" refers to an oligonucleotide subunit containing a sugar group and a heterocyclic base, as well as analogues of such subunits.

[0022] The terms “nucleoside” and “nucleotide” are intended to include not only known purine and pyrimidine bases (e.g., adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U)) but also other modified heterocyclic bases. Pyrimidines, purines, heterocyclic bases, and modified heterocyclic bases are referred to herein as “nucleic acid bases.” Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated heterocycles, or other heterocycles. Examples of such modifications include the addition of protecting groups such as diaminopurines and their derivatives, inosine and its derivatives, alkylated purines or pyrimidines, acylated purines or pyrimidines, thiolated purines or pyrimidines, or acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl, phenoxyacetyl, dimethylformamidine, dibutylformamidine, and N,N-diphenylcarbamate. In addition, the terms "nucleoside" and "nucleotide" include moieties that contain not only the conventional ribose and deoxyribose sugars but also other sugars. Modified nucleosides or nucleotides may also include modifications to the sugar moiety, for example, in which one or more hydroxyl groups are substituted with halogen atoms or aliphatic groups, or functionalized as ethers, amines, etc. Modified nucleosides or nucleotides also include modifications to internucleotide linkages or the skeletal moiety."Analog" refers to a molecule having structural features recognized in the literature as mimetic, a derivative having a similar structure, or other similar terminology, and includes, for example, polynucleotides incorporating non-natural (not normally occurring in nature) nucleotides, unnatural nucleotide mimetics such as 2'-modified nucleosides (e.g., 2'-MOE, 2'-OMe, 2'-F, 2'-cEt, LNA, UNA, etc.), peptide oligonucleotides, oligomeric nucleoside phosphonates, phosphorothioates, and any polynucleotides with substituents such as protecting or linking groups.

[0023] The term "oligonucleotide" refers to a compound containing multiple nucleoside subunits linked by internucleotide bonds. Therefore, the term also refers to a compound containing multiple nucleotide subunits or residues. Oligonucleotides may contain ribonucleosides, deoxyribonucleosides, or mixtures thereof. Oligonucleotides may comprise natural and / or unnatural nucleosides, nucleoside analogs, and modified nucleosides, and may also comprise natural (phosphate diester) and / or unnatural internucleotide bonds (e.g., amide bonds such as phosphorothioates, phosphonates, boranophosphonates, phosphoramidates, and peptide nucleic acids (PNA)).

[0024] In some embodiments, oligonucleotides may be named according to the nomenclature [number]-mer, where [number] indicates the number of nucleosides linked to each other by phosphate bonds. For example, a "2-mer," or dimer, refers to a compound having two nucleosides linked to each other by a single phosphate bond. A "5-mer" refers to five nucleosides linked to each other by four phosphate bonds.

[0025] The term “connected” means that two or more components are fluidly connected, physically connected, or both. The term “fluidically connected” means that two components are fluidly connected and include direct connections between the two components, and indirect connections in which one or more other components are in the flow path between the two components. For example, a first component and a second component are fluidly connected if the outlet of the first component is physically connected to the inlet of the second component, or if a conduit connects the first and second components, or if one or more intervening components, such as a valve, pump, or other structure, are between the two components as the fluid flows from the first component to the second component or vice versa.

[0026] As used herein, "mass peak" or "mass spectrometry signal" refers to a peak shown in the resulting spectrum or chromatographic peak (e.g., extracted ion chromatogram (EIC) or total ion chromatogram (TIC)) when a sample is analyzed using an MS instrument.

[0027] As used herein, “introduce” means to inject, fluidize, pour, alter, or otherwise add a sample to an MS instrument in any appropriate manner.

[0028] It is conceivable that similar or equivalent methods and materials to those described herein may be used in the practice of these instructions. Some exemplary methods and materials are described below.

[0029] The present invention and its composition are directed toward evaluating the performance of an MS instrument or system. In some embodiments, this evaluation may consist of or be part of the qualification of the MS instrument. In some embodiments, this evaluation may include a step of calibrating the mass axis of the mass spectrometer.

[0030] This method and standard composition are for mass spectrometry (MS) instruments. An MS instrument may include a detector configured to analyze a sample by measuring the mass-to-charge (m / z) ratio of ions received by the detector. The results may be presented as a mass spectrum, a plot of intensity as a function of mass-to-charge ratio, etc. Mass spectrometry is useful for identifying components of complex mixtures. An MS instrument may be any apparatus configured to separate, classify, or filter sample ions based on their respective masses (e.g., mass-to-charge ratio, i.e., m / z ratio). Examples of MS instruments include, but are not limited to, multipolar electrode structures (e.g., mass filters, ion traps), time-of-flight (TOF) analyzers, single quadrupole (SQ) LC / MSD, triple quadrupole (TQ or QQQ) LC / MS, quadrupole time-of-flight (TOF / Q-TOF), electrostatic analyzers (ESA), and magnetic field sectors.

[0031] The MS system 100 may generally include, in the order of the ion processing flow, an ion source 104, a mass spectrometer 108 downstream of the ion source 104, and an ion detector 112 positioned to receive ions from the mass spectrometer 108. From the viewpoint of Figure 1, the MS system 100 partitions the channels for ions and gas molecules, passing through the above-mentioned apparatus in a continuous direction from left to right, as indicated by the horizontal arrows. The MS system 100 also includes a vacuum system (not shown) for maintaining various internal regions of the MS system 100 at a controlled, lower-than-atmospheric pressure level. The vacuum system may include vacuum lines that communicate with the various internal regions via vacuum ports or exhaust ports, one or more vacuum generating pumps, and associated components. The vacuum lines may also remove non-analytical neutral molecules from the ion passages of the MS system 100. For simplicity, additional ion processing equipment, ion optics, electronics, and other hardware that may be included in the MS system 100 are not shown. For example, in some embodiments, the MS system 100 may include an ion mobility analysis stage.

[0032] The ion source 104 may be any type of continuous-beam or pulsed ion source suitable for generating sample ions for spectroscopic analysis, as will be recognized by those skilled in the art. Depending on the type of ionization performed, the ion source 104 may operate in a vacuum or at or near atmospheric pressure. Examples of ion sources include, but are not limited to, electron ionization (EI) sources, chemical ionization (CI) sources, photoionization (PI) sources, electrospray ionization (ESI) sources, atmospheric pressure chemical ionization (APCI) sources, atmospheric pressure photoionization (APPI) sources, field ionization (FI) sources, plasma or corona discharge sources, laser desorption ionization (LDI) sources, and matrix-assisted laser desorption ionization (MALDI) sources. The sample to be analyzed may be introduced into the ion source 104 by any suitable means, including a hyphenated technique in which the sample material is output 116 from analytical separation equipment such as gas chromatography (GC), a sample or standard injection pump, or (as will be discussed in more detail below) liquid chromatography (LC) equipment. In addition, the sample may be piped or pumped directly to the ion source of the mass spectrometer without the need for any other analytical equipment.

[0033] In some embodiments of this disclosure, at least one mass spectrometer (e.g., mass spectrometer 108) is based on a quadrupole mass filter or a linear ion trap, as further described below. The ion detector 112 may be any device configured to collect and measure the flow (or stream) of mass-identified ions output from the mass spectrometer 108. Examples of ion detectors include, but are not limited to, multichannel detectors (e.g., microchannel plate (MCP) detectors), electron multiplier tubes, photomultiplier tubes, image current detectors, and Faraday cups.

[0034] In a typical operation, the sample is introduced into the ion source 104, which generates ions from ionizable compounds in the sample and sends them to the mass spectrometer 108. The mass spectrometer 108 selectively sends the ions to the ion detector 112 based on the mass-to-charge (m / z) ratio. The mechanism of mass selection or filtering depends on the type of mass spectrometer. The ion detector 112 receives the ions and generates an ion measurement signal, from which the mass spectrum of the sample is constructed.

[0035] As also shown in Figure 1, in some embodiments, the MS system 100 may be configured to perform tandem MS (MS / MS). For example, the MS system 100 may be configured as a QqQ, qTOF, or QqTOF, TQTOF instrument. Thus, the MS system 100 may include a first mass spectrometer 120 upstream of the mass spectrometer 108 (the second or last mass spectrometer in such embodiments), and an ion fragmentation device such as a collision cell 124 between the first mass spectrometer 120 and the second mass spectrometer 108. The first mass spectrometer 120 is configured to select precursor ions of a specific m / z ratio or m / z ratio range and is typically (but not always) configured as a quadrupole mass filter. The collision cell 124 typically includes an RF-only, non-mass-separating ion guide enclosed within the cell. The cell is pressurized with an inert gas to a level sufficient to generate fragment ions from precursor ions by collision-induced dissociation (CID), as those skilled in the art will understand. However, fragmentation devices other than CID-based devices may be used, such as devices configured to perform electron capture dissociation (ECD), electron transfer dissociation (ETD), or infrared multiphoton dissociation (IRMPD). The second mass spectrometer 108 then separates the fragment ions based on their mass (m / z ratio) and sends the mass-separated fragment ions to the ion detector 112, which outputs a measurement signal, from which a mass spectrum is generated. In the tandem MS embodiment, both the first mass spectrometer 120 and the second mass spectrometer 108 may be based on a linear quadrupole electrode structure. Alternatively, the second mass spectrometer 108 may be another type of mass spectrometer, such as a three-dimensional pole trap, a time-of-flight (TOF) analyzer, an electrostatic trap, an electrostatic and / or magnetic field sector instrument, or an ion cyclotron resonance (ICR) cell (FT-ICR or FTMS).

[0036] As further shown in Figure 1, ion optics 128, 132, 136, and 140 may precede or follow one or more ion processing devices (e.g., a first mass spectrometer 120, a collision cell 124, a second mass spectrometer 108, or other ion guides not specifically shown). Ion optics 128, 132, 136, and 140 may include various types of lens elements, such as aperture lenses (centered on-axis ring electrodes, plane electrodes with centered on-axis apertures, split-plate or split-cylinder electrodes with centered on-axis open slots or gaps), parallel plane electrodes, and multipole rod electrodes. Any of the ion optics 128, 132, 136, and 140 may be or include field terminators as described herein.

[0037] The MS system 100 may also include a computing device (or system controller) 144. The computing device 144 is schematically depicted as one or more modules (or units or components) configured to control, monitor, and / or time the various functional aspects of the MS system 100 described above. One or more modules of the computing device 144 may be, or be embodied in, a desktop computer, a laptop computer, a portable computer, a tablet computer, a handheld computer, a mobile computing device, a personal digital assistant (PDA), a smartphone, etc. The computing device 144 may also schematically depict all voltage sources, which are not specifically shown, and timing controllers, clocks, frequency / waveform generators, etc., necessary for applying voltage to the various components of the MS system 100. The computing device 144 may also be configured to receive ion detection signals from the ion detector 112 and to perform data acquisition and signal analysis tasks as necessary to generate chromatograms, drift spectra, and mass (m / z ratio) spectra characterizing the sample under analysis. The computing device 144 may also be configured to provide and control a user interface that provides a screen display of spectroscopic analysis data and other data that the user can interact with. The computing device 144 may include one or more readers into which a substantial computer-readable (machine-readable) medium containing instructions for performing all or part of any of the methods disclosed herein can be inserted. For all these purposes, the computing device 144 may signal to various components of the MS system 100 via a wired or wireless link (partially indicated, for example, by a dashed line between the computing device 144 and the ion detector 112). Also for these purposes, the computing device 144 may include one or more types of hardware, firmware and / or software, as well as one or more processors, memory and databases.

[0038] As described above, the MS system 100 may include at least one quadrupole ion guide assembly, which includes a quadrupole ion guide and at least one field terminator. The quadrupole ion guide may be any of the ion processing devices described above, which are configured as a linear quadrupole electrode structure and are preceded and / or followed by a field terminator. In one non-limiting example, the quadrupole ion guide assembly may include a first mass spectrometer 120 (e.g., a mass filter), a preceding field terminator (pre-filter) schematically depicted as an ion optical system 128, and / or a succeeding field terminator (post-filter) schematically depicted as an ion optical system 132.

[0039] MS equipment may include, or be part of, a system comprising more than one mass spectrometer, particularly when ion fragmentation is desired. For example, the mass spectrometer may include a mass filter, followed by a collision cell or other ion fragmentation device, and then another mass filter or analyzer.

[0040] MS equipment may be part of an analytical system that includes other equipment such as chromatography equipment. MS equipment may be configured for direct injection mass spectrometry, liquid chromatography / mass spectrometry, gas chromatography / mass spectrometry, ion mobility / mass spectrometry, supercritical fluid chromatography / mass spectrometry, or any combination thereof. In some embodiments, MS analysis is performed by liquid chromatography / mass spectrometry (LC / MS) or ultra-high pressure liquid chromatography / mass spectrometry (UHPLC / MS).

[0041] In some embodiments, the MS consists only of a sample pump that feeds the sample to the MS system's supply source or is co-discharged with the (U)HPLC wastewater.

[0042] In some embodiments, the method and system also include a liquid chromatography apparatus fluidically connected to the MS apparatus. Liquid chromatography includes, but is not limited to, normal-phase liquid chromatography (NPLC), hydrophilic interaction liquid chromatography (HILIC), reversed-phase liquid chromatography (RPLC) including ion-pairing reversed-phase liquid chromatography (IP-RPLC), high-performance liquid chromatography (HPLC), highly turbulent liquid chromatography (HTLC), and ultrahigh-performance liquid chromatography (UPLC).

[0043] In some embodiments, liquid chromatography includes hydrophilic interaction liquid chromatography (HILIC). HILIC can be used to separate small polar compounds on a polar stationary phase. In HILIC, a hydrophilic stationary phase is used with a reverse-phase eluent to elute the sample in order of increasing polarity. Suitable mobile phases for HILIC include acetonitrile (ACN) and other aprotic solvents that are miscible with water. Ionic additives, such as ammonium salts, can be used to adjust the mobile phase pH and ionic strength.

[0044] In some embodiments, the liquid chromatography comprises ion-pairing reversed-phase liquid chromatography (IP-RPLC). IP-RPLC is a technique for separating organic ions and partially ionized organic samples. In IP-RPLC, the same type of stationary and mobile phases are used as RPLC, with the ion-pairing reagent added to the mobile phase. The ion-pairing reagent may be, for example, an alkyl sulfonate, alkyl ester, or alkyl ammonium salt, thereby changing the retention time of the ionic sample. In some embodiments, the mobile phase buffer comprises hexafluoroisopropanol (HFIP) or triethylamine (TEA).

[0045] The method may include the step of introducing one or more samples into the MS instrument simultaneously with or separately from the standard (before or after). In some embodiments, the standard is introduced into the MS instrument simultaneously with the sample under consideration, and the known mass of the standard is used to dynamically or continuously correct or adjust the m / z calibration of the MS instrument. In some embodiments, the method includes the step of mixing the standard and the sample to form a mixture. The mixing step may be performed before introducing the mixture into the MS instrument, or the mixing step may be performed within the MS instrument by combining the delivery of separate components in a tank, or by combining the flow of each component through the use of a T-junction.

[0046] This method uses a standard that is suitable for introduction into a mass spectrometer and comprises one or more oligonucleotides. Examples of oligonucleotides include formulas I, II, III, or IV: X 1 -3'-PL-3'-X 2 (I) X 1 -5'-PL-5'-X 2 (II) X 1 -3'-PL-5'-X 2 (III) X 1 -5'-PL-3'-X 2 (IV) The compound in question is included, where PL is a phosphate bond. The phosphate bond may be -PO4-, -PO3S-, -PO3BH2-, -PO3-O-PO3-, or -PO3-O-PO2-O-PO3-. 1 and X 2 X may be a nucleoside, nucleotide, or oligonucleotide. 1 and X 2 X may be the same (e.g., deoxythymidine) or different (deoxythymidine and deoxycytidine). 1 -3'- is X 1This suggests that the 3'O- of the sugar group is attached to the phosphate bond. In other words, X 1 -3'- and -3'-X 2 The 3'O- group inside is X 1 and X 2 It is part of the -PO4- group between them. Similarly, -3'-X 2 X 2 This suggests that the 3'O- of the sugar group is attached to the phosphate bond. X in Equation II. 1 -5'- is X 1 This suggests that the 5'O- of the sugar group is attached to the phosphate bond, and -5'-X 2 X 2 This suggests that the 5'O- of the sugar group is attached to the phosphate bond. Similar bonds can be found in formulas III and IV.

[0047] In some embodiments of the present invention, the standard oligonucleotides are present in solution, or they are solubilized from a solid. Any oligonucleotide in the standard may have a length of 2 to 100 nucleic acid bases. In some embodiments, one or more (or all) oligonucleotides have a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleic acid bases. In some embodiments, one or more (or all) oligonucleotides have a length of 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, or 95-100 nucleic acid bases.

[0048] In some embodiments, the oligonucleotides in the standard are MOE, 2'-fluoro, 2'-OMe, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5-propynylcytidine, C-5-propynyluridine, 2-aminoadenosine, C-5-bromouridine, C-5-fluorouridine, C-5-iodouridine, C-5-propynyluridine, C-5-propynylcytidine, C-5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxy The compounds comprise soadenosine, 8-oxoguanosine, O(6)methylguanine, 2-thiocytidine, methylated bases, inserted bases, modified sugars (e.g., locked nucleic acid (LNA), unlocked nucleic acid (UNA), 2',4'-restricted 2'-O-ethyl c(Et), 2',4'-restricted 2'-O-methoxyethyl (cMOE), 2'-fluororibose (2'-F), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-O-MOE), ribose, 2'-deoxyribose, arabinose, and hexose, or any combination thereof).

[0049] In some embodiments, one or more oligonucleotides in the standard further comprise alkyl spacers such as dodecyl, undecyl, decyl, nonyl, octyl, heptyl, hexyl, pentyl, butyl, propyl, ethyl, and methyl. In some embodiments, oligonucleotides in the standard comprise GalNAc or PEG residues. In some embodiments, oligonucleotides in the standard comprise 2', 3', and / or 5' synthetic protecting groups such as tert-butyldimethylsilyl (TBDMS), methoxymethyl ether (MOM), triisopropylsilyloxymethyl (TOM), methoxyethoxymethyl ether (MEM), dimethoxytrityl (DMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl, ether, silyl ether, methyl ester, thiomorpholine-4-carbothioate (TC), bis(2-acetoxyethyl-oxy)methyl (ACE), and trityl. In some embodiments, one or more oligonucleotides in the standard are equipped with a nucleic acid base protecting group (e.g., acetyl, isobutyl, t-butylphenoxyacetyl (TAC), dimethylformamidine (dmf), or a benzoyl group). In some embodiments, one or more oligonucleotides in the standard are equipped with a phosphate having a cyanoethyl (CNET) protecting group.

[0050] In aspects of this disclosure, a standard (also called a standard composition) is supplied for mass spectrometry analysis and comprises at least one oligonucleotide in a known amount in a solvent capable of solubilizing a compound, and any other components that may be present in the composition. In some embodiments, the oligonucleotide may be used to calibrate a mass spectrometer operating in positive or negative ionization mode. In some embodiments, the oligonucleotide may be used to calibrate a mass spectrometer having an atmospheric pressure chemical ionization (APCI) mass spectrometer or a mass spectrometer having an electrospray (ESI) ion source. In some embodiments, the oligonucleotide may be selected to enable calibration over a wide mass range and to provide a reference mass peak in both positive and negative ionization modes. In various aspects, substantially the same components can be used for both APCI and electrospray mass spectrometry. To provide calibration over a wide mass range, the oligonucleotide may comprise a mixture of different distinct oligonucleotides, as in some embodiments of this disclosure. In embodiments, the calibration provides a linear or substantially linear mass calibration over the entire mass range being analyzed.

[0051] Tuning is the process of adjusting parameters to optimize mass accuracy and resolution, and to monitor the signal intensity (sensitivity) of the tuned ion. Calibration is the process of assigning accurate masses based on standard compounds of known masses.

[0052] The compositions, kits, and methods described herein offer various advantages over other compounds and methods that have been used to evaluate the performance of MS instruments. For example, according to some embodiments of this disclosure, the oligonucleotides provided herein can result in better accuracy, sensitivity, and consistency. The oligonucleotides disclosed herein, as well as the compositions, kits, and methods comprising them, result in improved ionization, mass signal, and sensitivity in negative-mode MS analysis.

[0053] In some embodiments, the standard structure is:

[0054] [ka]

[0055] It comprises an oligonucleotide having the following properties.

[0056] The inventors have found that such compounds are particularly suitable for use as standards for MS instruments in negative ionization mode. Without being bound by theory, such compounds are particularly suitable because the single central phosphate molecule produces a particularly distinct peak in the spectrum resulting from mass spectrometry analysis. Furthermore, thymine nucleic acid bases do not require further processing before being introduced into the MS instrument. Similar oligonucleotides containing other nucleic acid bases may be used, but some of these may require further processing, such as deprotection of nucleic acid bases, before introduction to protect the other nucleic acid base groups.

[0057] Examples of oligonucleotides include deoxythymidine-3'-PL-3'-deoxythymidine, deoxythymidine-5'-PL-5'-deoxythymidine, deoxyuridine-3'-PL-3'-deoxyuridine, deoxyuridine-5'-PL-5'-deoxyuridine, thymine-3'-PL-3'-thymine, thymine-5'-PL-5'-thymine, uridine-3'-PL-3'-uridine, uridine-5'-PL-5'-uridine, deoxythymidine-3'-PL-3'-deoxyuridine, deoxyuridine Xythymidine-5'-PL-5'-deoxyuridine, deoxythymidine-3'-PL-3'-thymidine, deoxythymidine-5'-PL-5'-thymidine, deoxythymidine-3'-PL-3'-deoxycytidine, deoxythymidine-5'-PL-5'-deoxycytidine, deoxycytidine-3'-PL-3'-deoxycytidine, deoxycytidine-5'-PL-5'-deoxycytidine, deoxyadenosine-3'-PL-3'-deoxyadenosine, deoxyadenosine-5'-PL- 5'-Deoxyadenosine, Deoxyguanosine-3'-PL-3'-Deoxyguanosine, Deoxyguanosine-5'-PL-5'-Deoxyguanosine, as well as MOE, 2'Fluoro, 2'OMe, 2-Aminoadenosine, 2-Thiothymidine, Inosine, Pyrrolo-Pyrimidine, 3-Methyladenosine, 5-Methylcytidine, C-5Propynylcytidine, C-5Propynyluridine, 2-Aminoadenosine, C-5Bromouridine, C-5Fluorouridine, C-5Iodouridine, C- This includes, but is not limited to, nucleotides comprising 5-propynyluridine, C-5-propynylcytidine, C-5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, inserted bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), or any combination thereof. Further examples of oligonucleotides include:

[0058] [ka]

[0059] This includes, but is not limited to, the following:

[0060] In some embodiments of the present invention and its composition, the standard composition is a solution. The solution may be an aqueous solution or a non-aqueous solution. The standard composition may contain one or more oligonucleotides in the solvent or diluent. In some embodiments, the solvent or diluent may be aqueous or an organic solvent. Examples of solvents or diluents include, but are not limited to, water, acetonitrile, methanol, ethanol, propanol, isopropanol, butanol, dichloromethane, dimethylformamide, N-methyl-2-pyrrolidone, acetone, toluene, benzene, chloroform, dimethylformamide, organic acids (e.g., acetic acid), amines (e.g., dimethylamine), or combinations thereof (acetonitrile and water, acetone and water, ethanol and water, methanol and water, formic acid diluted with water, acetic acid diluted with water, or ammonium hydroxide diluted with water). Any suitable solvent or diluent may be used.

[0061] The evaluation of mass spectrometry results includes the step of identifying one or more “mass peaks” or “mass spectrometry signals” corresponding to oligonucleotides in the resulting spectral EIC or TIC. The identified (or multiple) m / z peaks corresponding to the oligonucleotide standard are used when the amount is changed through concentration or injection volume. Linear, binomial, logarithmic, or other statistically related metric trend line results are assessed for comparability to a predetermined approval standard. The amount of change in linearity or other regression models contributes to r-squared analysis. The method of this disclosure is used with deoxythymidine-3'-PL-5'-deoxythymidine to assess the R of a trend line greater than a predetermined approval standard (e.g., 0.900, 0.950, 0.990, 0.995, or 0.999). 2 A linearity result satisfying the value was generated.

[0062] In some embodiments, the method includes the steps of determining the difference between an expected intensity for one or more oligonucleotides in an oligonucleotide and the corresponding actual intensity for one or more oligonucleotides in a mass spectrum, wherein the expected intensity is determined from a series of known different concentration injections or assessments of one or more oligonucleotides, and adjusting the mass spectrometer as part of the qualification or calibration of the mass spectrometer based on the difference between the expected intensity and the actual intensity in the mass spectrum.

[0063] In another aspect, the present disclosure provides a method for calibrating a mass spectrometer. In some embodiments, the method includes a) obtaining a mass spectrum of at least one standard composition comprising a plurality of oligonucleotides, or a kit comprising at least one oligonucleotide, according to any aspect and embodiment described herein; b) determining the difference between the expected mass peaks for the plurality of oligonucleotides in the standard composition and the corresponding actual mass peaks in the mass spectrum; and c) adjusting the mass spectrometer based on the difference between the expected mass peaks and the actual mass peaks.

[0064] The present invention can be used to calibrate, standardize, or qualify the MS / MS mode of an MS instrument using mass spectrometry signals from standard evaluations disclosed herein.

[0065] The method may further include the step of adding a standard to a standard container. The standard is introduced into the MS instrument by flowing from the standard container. In some embodiments, the standard is continuously flowed into the MS instrument for a selected or predetermined period of time.

[0066] In another aspect, the method further includes the steps of connecting an MS instrument to an oligonucleotide synthesis reaction chamber and receiving a sample from the oligonucleotide synthesis reaction chamber. Oligonucleotide synthesis reaction chambers and methods are discussed in Patent Documents 1, 2, 3, and 4.

[0067] In some embodiments, the standard is introduced into the MS instrument in a continuous manner during the analysis of the sample from the oligonucleotide synthesis reaction chamber. The sample from the oligonucleotide synthesis reaction chamber may comprise oligonucleotides having synthetic protecting groups such as acetyl, isobutyl, TAC, dmf, benzoyl, TBDMS, MOM, MEM, DMT, PMB, methylthiomethyl ether, pivaloyl, ether, silyl ether, methyl ester, and trityl. In some embodiments, the sample from the oligonucleotide synthesis reaction chamber comprises oligonucleotides having phosphates with a CNET protecting group. In some embodiments, the sample from the oligonucleotide synthesis reaction chamber comprises oligonucleotides having GalNAc and / or PEG residues.

[0068] This disclosure includes formulas I to IV: X 1 3'-PL-3'-X 2 (I) X 1 5'-PL-5'-X 2 (II) X 1 3'-PL-5'-X 2 (III) X 1 5'-PL-3'-X 2 (IV) (In the formula, X 1 , X 2We also provide standard compositions comprising multiple oligonucleotides having the structure (and PL having the meanings described above). In some embodiments, the standard comprises two or more, three or more, four or more, or more number of oligonucleotides having the structures of formulas I to IV. In some embodiments, each oligonucleotide in the standard has a different length and / or a different molecular weight. For example, the standard may comprise two oligonucleotides having the structure of formula I, in which case the oligonucleotides have different lengths and / or different molecular weights. In some embodiments, the standard may be a solution or a solid. In solution may mean an aqueous solution or a non-aqueous solution. Examples Example 1 3'-O-(5'-thymidyl)thymidine was prepared by solid-phase synthesis using an inverted dT CPG synthesis support. The solid synthesis support has a 3'-terminal dimethoxytrityl protection, which is removed with dichloroacetic acid. Deoxythymidine phosphoramidite was conjugated to the inverted dT introduced into the support by conventional oligonucleotide synthesis, by co-delivering the amidite with 5-ethylthiotetrazole (ETT), recirculating the charged ETT and phosphoramidite solution, and then removing the terminal DMT protecting group of the charged phosphoramidite with DCA. Following conjugation, diethylamine washing was performed, followed by a cleavage / deprotection step using ammonium hydroxide, and ammonia was evaporated using a rotovap until the pH reached <10.

[0069] Next, the synthesized 2-mer was tested for purity. The synthesized product was subjected to a 2.1 × 100 mm, 1.7 μm Waters Acquity UPLC BEH test. TMThe mixture was introduced into a Shield RP18 column. The first mobile phase (mobile phase A) consisted of 90% 10mM ammonium acetate and 10% methanol at pH 8.0, making up 85% of the mixture. The second mobile phase (mobile phase B) consisted of 50% ultrapure water and 50% methanol, making up 15% of the mixture. Figure 2 shows the chromatogram of the synthesized product. A purity of 96.8% was obtained.

[0070] Oligonucleotide samples were prepared at concentrations of 1 OD / mL (optical density per milliliter), 5 OD / mL, and 10 OD / mL. 4 μL of each sample was injected into an MS apparatus at a flow rate of 0.3 mL / min for 5 minutes at a column temperature of 40 degrees Celsius. Example 2 The linearity test featured a mobile phase of 20 mM ammonium formate in a 75 / 25 mixture of methanol and water. Oligonucleotide samples were prepared at a concentration of 0.1 OD / mL. Volumes of 1, 3, 5, 7, 10, 15, and 20 μL were injected into MS apparatus at a flow rate of 0.3 mL / min and a runtime of 5 minutes for separate analysis. The MS results are shown in Table 1.

[0071] [Table 1]

[0072] These results were refined by incorporating extracted ion chromatograms (EICs) at 545.1 m / z and comparing them with the amounts of oligonucleotides used in each analysis, thereby generating linearity curves. Figure 3 shows the linearity curves calculated via linear regression, demonstrating that sample concentrations of 0.1 OD / mL injected over a volume range of 1–20 μL produced an acceptable trend response from the MS instrument.

[0073] Next, this process of the linearity test was repeated by injecting 1, 3, 5, 7, 10, 15, and 20 μL of 1.0 OD / mL 2-mer specimens. The MS results are shown in Table 2.

[0074] [Table 2]

[0075] These results were examined by incorporating the extracted ion chromatogram at 545.1 m / z and comparing it with the injected concentration to generate a linearity curve. Figure 4 shows the linearity curve calculated via binomial regression, demonstrating that a sample concentration of 1.0 OD / mL injected over a volume range of 1–20 μL produced an acceptable trend response from the MS instrument. Example 3 An oligonucleotide compound comprising five nucleotides (a 5-mer with a 3' bond at the 3' terminus) was synthesized by repeating the synthesis steps described in Example 1. This oligonucleotide compound contains three charge states, i.e., m / z values ​​(485.3 (3rd), 728.1 (2nd), and 1457.0 (1st) m / z), which typically correspond to phosphate bonds in MS results.

[0076] The 5-mer sample was injected into an MS instrument under the conditions described in Example 1 and analyzed using the MS instrument. The MS instrument showed two known charge states for the 5-mer, which suggests good performance of the MS instrument and provides an opportunity to dynamically adjust its mass axis calibration based on the observed changes in the mass spectrum.

[0077] Example 4 Oligonucleotide compounds (2-mer) comprising two nucleotides were synthesized by repeating the synthesis steps described in Example 1. These oligonucleotide compounds can be injected into a mass spectrometer having a collision cell or other fragmentation capability enabling mass spectrometry / mass spectrometry (MS / MS) or tandem mass spectrometry. The theoretical MS / MS fragments of the inverted dT-dT standard are shown below.

[0078] [ka]

[0079] [ka]

[0080] The resulting fragment ions (125.0, 195.0, 293.0, 321.1, and 419.1 in the presented examples, with a parent ion m / z of 545.1 m / z) can be used to qualify the mass axis calibration and linearity of the MS / MS system. This is demonstrated by the results shown in Table 3 and Figures 5-7.

[0081] [Table 3]

[0082] Exemplary Embodiments The exemplary embodiments presented relating to the subject matter disclosed herein include, but are not limited to, the following:

[0083] Embodiment 1 A method for evaluating the performance of a mass spectrometer, Steps include setting up a mass spectrometer (MS) and A step of introducing a standard to the MS instrument, wherein the standard is Formula I, Formula II, Formula III, or Formula IV: X 1 3'-PL-3'X 2 (I) X 1 5'-PL-5'X 2 (II) X 1 3'-PL-5'X 2 (III) X 1 5'-PL-3'X 2 (IV) It comprises an oligonucleotide having the following structure, During the ceremony, PL is a phosphate bond comprising -PO4-, -PO3-O-PO3-, -PO3S, -PO3BH2- or PO3-O-PO2-O-PO3-, X 1 and X 2 Steps are independently nucleosides, nucleotides, or oligonucleotides. A method comprising the step of evaluating one or more mass spectrometry signals corresponding to the oligonucleotide from the mass spectrometer.

[0084] Embodiment 2 The method of Embodiment 1, wherein the evaluation step includes identifying one or more mass peaks corresponding to the oligonucleotide, and comparing the identified one or more mass peaks with one or more expected mass peaks for the oligonucleotide.

[0085] Embodiment 3: The method of Embodiment 1 or Embodiment 2, further comprising the step of adjusting the MS instrument based on the evaluation of one or more mass peak signals.

[0086] Embodiment 4: The method according to any of Embodiments 1 to 3, wherein the mass spectrometer has a negative ionization mode, and the standard is introduced for negative mode mass spectrometry analysis.

[0087] Embodiment 5: The method of Embodiment 4, further comprising the step of qualifying the MS instrument for a negative ionization mode.

[0088] Embodiment 6 The method of Embodiment 5, wherein the qualification includes the steps of analyzing the oligonucleotide in a plurality of quantities, and determining whether the one or more mass spectrometry signals are substantially linear with respect to the plurality of quantities.

[0089] Embodiment 7: The method of Embodiment 6, wherein the analytical step is a nonlinear regression analysis on the plurality of quantities used to calibrate or qualify a mass spectrometer.

[0090] Embodiment 8: Any method of Embodiments 1 to 7, further comprising the step of performing mass axis calibration.

[0091] Embodiment 9: The method of any of Embodiments 1 to 8, wherein the standard is a continuous reference standard that is introduced into the MS instrument in a continuous manner during sample analysis.

[0092] Embodiment 10: The method according to any of Embodiments 1 to 9, wherein the standard is an internal standard introduced into the MS instrument simultaneously with the sample for analysis.

[0093] Embodiment 11X 1 and X 2 The method is one of Embodiments 1 to 10, wherein the nucleoside is the same.

[0094] Embodiment 12 X 1 and X 2 The method of any of Embodiments 1 to 10, wherein is a different nucleoside.

[0095] Embodiment 13 The method according to any of Embodiments 1 to 10, wherein the oligonucleotide of formula I is deoxythymidine-3'-PL-3'-deoxythymidine.

[0096] Embodiment 14: The method according to any of Embodiments 1 to 13, wherein the oligonucleotide is present in solution.

[0097] Embodiment 15: The method according to any of Embodiments 1 to 14, wherein the oligonucleotide is solubilized from a solid.

[0098] Embodiment 16 The oligonucleotide is the length of 2 to 100 nucleic acid bases, according to any of Embodiments 1 to 15.

[0099] Embodiment 17 The method of any of Embodiments 1 to 16, wherein the oligonucleotide has a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleic acid bases.

[0100] Embodiment 18 The oligonucleotide is a nucleic acid base of length 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, or 95-100, according to any of Embodiments 1 to 16.

[0101] Embodiment 19 The oligonucleotides are MOE, 2'fluoro, 2'Ome, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C-5 bromouridine, C-5 fluorouridine, C-5 iodouridine, C-5 propynyluridine, C-5 propynylcytidine, C-5 methylcytidine, 2-aminoadenosine A method according to any one of Embodiments 1 to 18, comprising a nucleotide comprising noadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, LNA, UNA, C(et), 2-thiocytidine, methylated base, inserted base, modified sugar (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), or any combination thereof.

[0102] Embodiment 20 The method of Embodiment 1, wherein the oligonucleotide further comprises TBDMS, MOM, MEM, DMT, TOM, ACE, TC, PMB, methylthiomethyl phosphate, ether, pivaloyl, ether, silyl ether, methyl ester, and trityl.

[0103] Embodiment 21: The method according to any of Embodiments 1 to 20, wherein the oligonucleotide further comprises an alkyl spacer.

[0104] Embodiment 22: The method of Embodiment 21, wherein the alkyl spacer is selected from dodecyl, undecyl, decyl, nonyl, octyl, heptyl, hexyl, pentyl, butyl, propyl, ethyl, and methyl.

[0105] Embodiment 23 The method of any of Embodiments 1 to 22, wherein the oligonucleotide further comprises a GalNAc and / or PEG residue.

[0106] Embodiment 24 The method of any of Embodiments 1 to 23, wherein the oligonucleotide further comprises 2', 3', and / or 5' synthetic protecting groups.

[0107] Embodiment 25 The method of Embodiment 24, wherein the 2', 3' and / or 5' synthetic protecting groups are selected from TBDMS, MOM, MEM, TOM, ACE, TC, DMT, PMB, methylthiomethyl ether, pivaloyl, ether, silyl ether, methyl ester, and trityl.

[0108] Embodiment 26: The method according to any of Embodiments 1 to 25, wherein the oligonucleotide is composed of 3' and / or 5' bonds.

[0109] Embodiment 27: The method according to any of Embodiments 1 to 26, wherein the oligonucleotide is provided with a synthetic protecting group.

[0110] Embodiment 28: The method of Embodiment 27, wherein the synthetic protecting group is selected from the group consisting of acetyl, isobutyl, TAC, dmf, and benzoyl.

[0111] Embodiment 29: The method according to any one of Embodiments 1 to 28, wherein the oligonucleotide comprises a phosphate having a CNET protecting group.

[0112] Embodiment 30: Any method of Embodiments 1 to 29, further comprising the step of calibrating, standardizing, or qualifying the MS / MS mode of the MS instrument using the evaluated one or more mass spectrometry signals.

[0113] Embodiment 31: The method of Embodiment 30, wherein the standard is used to calibrate the MS / MS mode of a mass spectrometer.

[0114] Embodiment 32: The method of Embodiment 30, wherein the standard is used to standardize the MS / MS mode of the mass spectrometer.

[0115] Embodiment 33: The method of Embodiment 30, wherein the standard is used to qualify the MS / MS mode of the mass spectrometer.

[0116] Embodiment 34: The method of Embodiment 30, wherein the standard is injected into the MS instrument together with the sample, and the known mass of the standard is used to dynamically or continuously correct or adjust the m / z calibration of the MS instrument.

[0117] Embodiment 35 A method according to any of Embodiments 1 to 34, further comprising the step of putting the standard into a standard container, and introducing the standard into the MS apparatus by flowing it out of the standard container.

[0118] Embodiment 36: The method of Embodiment 35, wherein the standard is continuously flowed into the MS apparatus for a selected or predetermined period of time.

[0119] Embodiment 37 A method of any one of Embodiments 1 to 36, further comprising the steps of fluidly connecting the MS apparatus to an oligonucleotide synthesis reaction chamber and receiving a sample from the oligonucleotide synthesis reaction chamber.

[0120] Embodiment 38: The method of Embodiment 37, wherein the standard is introduced into the MS instrument in a continuous manner during the analysis of the sample from the oligonucleotide synthesis reaction chamber.

[0121] Embodiment 39: The method of Embodiment 37, wherein the sample from the oligonucleotide synthesis reaction chamber comprises an oligonucleotide having a synthetic protecting group.

[0122] Embodiment 40 The method of Embodiment 39, wherein the synthetic protecting group is selected from acetyl, isobutyl, TAC, dmf, benzoyl, TBDMS, MOM, MEM, DMT, PMB, methylthiomethyl ether, pivaloyl, ether, silyl ether, methyl ester, and trityl.

[0123] Embodiment 41 The method of Embodiment 37, wherein the sample from the oligonucleotide synthesis reaction chamber comprises an oligonucleotide having a phosphate with a CNET protecting group.

[0124] Embodiment 42 The method of Embodiment 37, wherein the sample from the oligonucleotide synthesis reaction chamber comprises an oligonucleotide comprising a GalNAc and / or PEG residue.

[0125] Embodiment 43 The method according to any of Embodiments 1 to 42, wherein the standard oligonucleotide comprises a plurality of oligonucleotides having the structure of formula I, II, III, or IV.

[0126] Embodiment 44: The method of Embodiment 43, wherein the two or more oligonucleotides have different molecular weights.

[0127] Embodiment 45: The method of Embodiment 43, wherein the two or more oligonucleotides have different lengths.

[0128] Please note that, in consideration of this disclosure, the methods and compositions can be carried out in accordance with this teaching. Furthermore, various components, materials, structures and parameters are included for illustrative purposes only and not in any limiting sense. In consideration of this disclosure, this teaching may be carried out in other uses, while remaining within the scope of the appended claims, and components, materials, structures and equipment for carrying out these uses may be determined.

Claims

1. A method for evaluating the performance of a mass spectrometer instrument, The steps include setting up a mass spectrometer (MS) and A step of introducing a standard to the MS instrument, wherein the standard is Formula I, Formula II, Formula III, or Formula IV: X １ 3´-PL-3´X ２ (I) X １ 5´-PL-5´X ２ (II) X １ 3´-PL-5´X ２ (III) X １ 5´-PL-3´X ２ (IV) It comprises an oligonucleotide having the following structure, During the ceremony, PL is, -PO ４ -, -PO ３ -O-PO ３ -, -PO ３ S, -PO ３ BH ２ - or -PO ３ -O-PO ２ -O-PO ３ a phosphate bond comprising - X １ and X ２ Steps are independently nucleosides, nucleotides, or oligonucleotides. A method comprising the step of evaluating one or more mass spectrometry signals corresponding to the oligonucleotide from the mass spectrometer.

2. The method according to claim 1, wherein the evaluation step includes identifying one or more mass peaks corresponding to the oligonucleotide, and comparing the identified one or more mass peaks with one or more expected mass peaks for the oligonucleotide.

3. The method according to claim 1, further comprising the step of adjusting the MS instrument based on the evaluation of the one or more mass peak signals.

4. The method according to claim 1, wherein the mass spectrometer has a negative ionization mode, and the standard is introduced for negative mode mass spectrometry analysis.

5. The method of claim 4, further comprising the step of qualifying the MS instrument for a negative ionization mode.

6. The aforementioned qualification is, A step of analyzing the oligonucleotide in multiple quantities, and The method according to claim 5, further comprising the step of determining whether the one or more mass spectrometry signals are substantially linear with respect to the plurality of quantities.

7. The method according to claim 6, wherein the analytical step is a nonlinear regression analysis on the plurality of quantities used to calibrate or qualify a mass spectrometer.

8. The method according to claim 1, further comprising the step of performing mass axis calibration.

9. The method according to claim 1, wherein the standard is a continuous reference standard that is introduced into the MS instrument in a continuous manner during sample analysis.

10. The method according to claim 1, wherein the standard is an internal standard introduced into the MS instrument simultaneously with the sample for analysis.

11. X １ and X ２ The method according to claim 1, wherein is the same nucleoside.

12. X １ and X ２ The method according to claim 1, wherein is a different nucleoside.

13. The method according to claim 1, wherein the oligonucleotide of formula I is deoxythymidine-3'-PL-3'-deoxythymidine.

14. The method according to claim 1, wherein the oligonucleotide is present in a solution.

15. The method according to claim 1, wherein the oligonucleotide is solubilized from a solid.

16. The method according to claim 1, wherein the oligonucleotide has a length of 2 to 100 nucleic acid bases.

17. The method according to claim 1, wherein the oligonucleotide has a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleic acid bases.

18. The method according to claim 1, wherein the oligonucleotide has a length of 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, and 95-100 nucleic acid bases.

19. The oligonucleotides are MOE, 2'-fluoro, 2'-Ome, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C-5 bromouridine, C-5 fluorouridine, C-5 iodouridine, C-5 propynyluridine, C-5 propynylcytidine, C-5 methylcytidine, 2 The method according to claim 1, comprising a nucleotide comprising aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, LNA, UNA, C (et), 2-thiocytidine, methylated base, inserted base, modified sugar (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), or any combination thereof.

20. The method according to claim 1, wherein the oligonucleotide further comprises TBDMS, MOM, MEM, DMT, TOM, ACE, TC, PMB, methylthiomethylphosphate, ether, pivaloyl, ether, silyl ether, methyl ester, and trityl.

21. The method according to claim 1, wherein the oligonucleotide further comprises an alkyl spacer.

22. The method according to claim 21, wherein the alkyl spacer is selected from dodecyl, undecyl, decyl, nonyl, octyl, heptyl, hexyl, pentyl, butyl, propyl, ethyl, and methyl.

23. The method according to claim 1, wherein the oligonucleotide further comprises a GalNAc and / or PEG residue.

24. The method according to claim 1, wherein the oligonucleotide further comprises 2', 3', and / or 5' synthetic protecting groups.

25. The method according to claim 24, wherein the 2', 3' and / or 5' synthetic protecting group is selected from TBDMS, MOM, MEM, TOM, ACE, TC, DMT, PMB, methylthiomethyl ether, pivaloyl, ether, silyl ether, methyl ester, and trityl.

26. The method according to claim 1, wherein the oligonucleotide is composed of 3' and / or 5' bonds.

27. The method according to claim 1, wherein the oligonucleotide is provided with a synthetic protecting group.

28. The method according to claim 27, wherein the synthetic protecting group is selected from the group consisting of acetyl, isobutyl, TAC, dmf, and benzoyl.

29. The method according to claim 1, wherein the oligonucleotide comprises a phosphate having a CNET protecting group.

30. The method according to claim 1, further comprising the step of calibrating, standardizing, or qualifying the MS / MS mode of the MS instrument using the evaluated one or more mass spectrometry signals.

31. The method according to claim 30, wherein the standard is used to calibrate the MS / MS mode of a mass spectrometer.

32. The method according to claim 30, wherein the standard is used to standardize the MS / MS mode of the mass spectrometer.

33. The method according to claim 30, wherein the standard is used to qualify the MS / MS mode of the mass spectrometer.

34. The method according to claim 30, wherein the standard is injected into the MS instrument together with the sample, and the known mass of the standard is used to dynamically or continuously correct or adjust the m / z calibration of the MS instrument.

35. The method according to claim 1, further comprising the step of putting the standard into a standard container, and introducing the standard into the MS apparatus by flowing it out of the standard container.

36. The method according to claim 35, wherein the standard is continuously flowed into the MS apparatus for a selected or predetermined period of time.

37. The method according to claim 1, further comprising the steps of: fluidly connecting the MS apparatus to an oligonucleotide synthesis reaction chamber; and receiving a sample from the oligonucleotide synthesis reaction chamber.

38. The method according to claim 37, wherein the standard is introduced into the MS instrument in a continuous manner during the analysis of the sample from the oligonucleotide synthesis reaction chamber.

39. The method according to claim 37, wherein the sample from the oligonucleotide synthesis reaction chamber comprises an oligonucleotide having a synthetic protecting group.

40. The method according to claim 39, wherein the synthetic protecting group is selected from acetyl, isobutyl, TAC, dmf, benzoyl, TBDMS, MOM, MEM, DMT, PMB, methylthiomethyl ether, pivaloyl, ether, silyl ether, methyl ester, and trityl.

41. The method according to claim 37, wherein the sample from the oligonucleotide synthesis reaction chamber comprises an oligonucleotide having a phosphate having a CNET protecting group.

42. The method according to claim 37, wherein the sample from the oligonucleotide synthesis reaction chamber comprises an oligonucleotide comprising a GalNAc and / or PEG residue.

43. The method according to claim 1, wherein the standard oligonucleotide comprises a plurality of oligonucleotides having the structure of formula I, II, III, or IV.

44. The method according to claim 43, wherein the two or more oligonucleotides have different molecular weights.

45. The method according to claim 43, wherein the two or more oligonucleotides have different lengths.

Images