Methods and systems for hydrophilic phase extraction

Abstract

Disclosed herein are embodiments of methods for oligonucleotide analysis using novel solid-phase extraction and hydrophilic interaction liquid chromatography.The unique polarity-based retention method provided herein provides high recovery extraction.The method improves assay reliability and reproducibility, and reaches picomolar sensitivity on accurate mass platforms, which is clearly beneficial.Systems and computer program products for implementing these methods are also disclosed herein.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 295,585, entitled "METHOD AND SYSTEM FOR ION-PAIR-FREE HYDROPHILIC PHASE EXTRACTION," filed December 31, 2021, which is incorporated by reference in its entirety.

[0002] (Technical field) The present disclosure relates generally to bioanalytical separation techniques and more particularly, but not exclusively, to methods and systems for hydrophilic phase extraction. [Background technology]

[0003] (background) In the field of oligonucleotide bioanalysis, there are many challenges for any quantitative platform, not limited to LC-MS. The selected means of biological sample preparation and extraction directly impacts the performance endpoint of the analytical platform used. Mainly, challenges may arise from the difficulty of using ion-pairing reagents and / or from using established extraction means that are traditionally designed for hydrophobic small molecules, but not polar and ionic biologics. Therefore, a novel method and system for analysis using hydrophilic phase extraction is provided herein. Summary of the Invention [Means for solving the problem]

[0004] (overview) Disclosed herein are methods embodiments for oligonucleotide analysis using solid phase extraction and hydrophilic interaction liquid chromatography methods.

[0005] In some embodiments, a method for determining the amount and / or presence of a compound of interest comprises the steps of: (i) providing a sample suspected of containing the compound of interest; (ii) performing an extraction of the compound of interest, which is a hydrophilic phase extraction; (iii) isolating the compound of interest from other components of the sample by liquid chromatography; and (iv) analyzing the compound of interest by mass spectrometry.

[0006] The system for carrying out the disclosed method is also described herein.In some embodiments, the system for determining the amount and / or presence of a compound of interest in a sample from a subject can include a solid phase extraction system, a liquid chromatography system, and a mass spectrometry detection system.

[0007] Computer program products are also described herein which, when implemented on one or more data processors, may cause the one or more data processors to perform operations that direct at least one of the following steps: providing a sample suspected of containing a compound of interest; performing solid phase extraction of the compound of interest from a biological matrix; isolating the compound of interest from other components of the sample by liquid chromatography; and analyzing the compound of interest by mass spectrometry. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows a flow chart for sample preparation and hydrophilic phase extraction according to an embodiment of the present disclosure.

[0009] [Diagram 2]FIG. 2 shows a chromatograph of hydrophilic interaction liquid chromatography according to an embodiment of the present disclosure, showing separation of the analyte GNV705 AS (center, 4.29 min) from the earlier eluting N-1 and N-2 metabolites (both at 4.1 min) and the later eluting candidate internal standard (4.4 min).

[0010] [Diagram 3] FIG. 3 shows a three-dimensional bar graph illustrating the LC-MS response comparison of the analyte GNV705 AS and an internal standard according to an embodiment of the present disclosure, where an initial plasma volume of 100 μl was diluted to various volumes before loading.

[0011] [Figure 4] FIG. 4 shows a bar graph of a dissolution volume optimization study according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description of the Invention The following description describes various aspects and embodiments of the present method and system. Any particular embodiment is not intended to define the scope of the present method and system. Rather, the embodiments merely provide non-limiting examples of various methods and systems that are at least included within the scope of the present method and system. This description should be read from the perspective of a person skilled in the art. Thus, information that is well known to a person skilled in the art is not necessarily included.

[0013] (definition) The present disclosure will now be described more fully hereinafter. The present disclosure can be embodied in many different forms, and the present disclosure should not be construed as being limited to the embodiments shown herein. Rather, these embodiments are provided so that the present disclosure will satisfy applicable legal requirements. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety. If the definitions set forth in this section are contrary to or otherwise inconsistent with the definitions set forth in the patents, applications, published applications and other publications incorporated herein by reference, the definitions set forth in this section shall prevail over the definitions incorporated herein by reference.

[0014] Illustrative examples are provided to orient the reader to the general subject matter discussed herein, and these illustrative examples are not intended to limit the scope of the disclosed concepts. The description of the figures in the detailed description describes various additional features and examples with reference to the drawings in which like numerals indicate like elements, and directional descriptions are used to describe illustrative aspects, but as with those illustrative aspects, those directional descriptions should not be used to limit the disclosure.

[0015] When introducing elements of the disclosure or embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that one or more of those elements are present. The terms "comprising," "including," and "having" are intended to be open-ended and mean that there may be additional elements other than the listed elements. It is understood that aspects and embodiments of the disclosure described herein include "consisting of" and / or "consisting essentially of" aspects and embodiments.

[0016] The term "and / or", when used in a list of two or more items, means that any one of the listed items may be used alone or in combination with any one or more of the listed items. For example, the phrase "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The phrase "A, B and / or C" is intended to mean A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0017] Various aspects of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Thus, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges within that range such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range (e.g., 1, 2, 3, 4, 5, and 6). This applies regardless of the breadth of the range.

[0018] The embodiments of the present invention may be used to analyze samples or biological samples. The samples may be in liquid, solid, and / or semi-solid form. The biological samples may include tissue, blood, biofluids, biosolids, etc., and combinations thereof. Thus, the term "biological sample" includes, by way of example, but is not limited to, whole blood, plasma or serum, urine, cerebrospinal fluid (CSF), lymph samples, saliva, sputum, fecal samples, lavage, semen, tissues, and / or body fluids, and chemical components thereof, in raw form and / or in preparations.

[0019] As used herein, the terms "compound of interest," "component of interest," or "biomarker of interest" are any marker that can provide biological information about the physiological state of an organism. In certain embodiments, the presence or absence of the biomarker can be informative. In other embodiments, the level of the biomarker can be informative. In certain embodiments, the component of interest can include an oligonucleotide. The oligonucleotide can be a therapeutic agent.

[0020] As used herein, the terms "subject" and "individual" may be used interchangeably. A subject may include an animal. Thus, in some embodiments, the sample is obtained from a mammal, including but not limited to a dog, cat, horse, rat, monkey, etc. In some embodiments, the sample is obtained from a human subject. In some embodiments, the subject is a patient, i.e., a living individual in a clinical setting for the diagnosis, prognosis, or treatment of a disease or condition.

[0021] As used herein, the terms "purify", "separate", "extract" or their derivatives do not necessarily refer to the removal of all substances other than the analyte of interest from a sample matrix. Instead, in some embodiments, the terms "purify", "separate", or "extract" refer to a procedure that enhances the amount of one or more analytes of interest relative to one or more other components present in the sample matrix. In some embodiments, the "purify", "separate", and / or "extract" procedures can be used to remove one or more sample components that may interfere with the detection of the biomarker of interest (e.g., one or more components that may interfere with the detection of the analyte by mass spectrometry).

[0022] As used herein, "chromatography" refers to a process in which a mixture of chemicals carried by a liquid or gas is separated into constituent components as a result of the differential distribution of their chemical entities as they flow around or over a liquid or solid stationary phase.

[0023] As used herein, "liquid chromatography" (LC) refers to the process of selective retardation of one or more components of a mobile solution as the fluid permeates through a column or capillary pathway of finely divided material. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid (i.e., the mobile phase) as the fluid moves relative to the stationary phase. "Liquid chromatography" includes reversed-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), and hydrophilic interaction liquid chromatography (HILIC). HILIC is a powerful chromatographic mode that involves a combination of electrostatic interactions and partitioning between a water-rich layer adsorbed on a polar stationary phase and a high acetonitrile mobile phase, in which retention generally increases with the polarity of the analyte.

[0024] As used herein, the term "analytical column" refers to a chromatographic column having sufficient chromatographic plates to effect separation of the components of a test sample matrix. Preferably, the components eluted from the analytical column are separated in such a way as to allow the presence or amount of the analyte of interest to be determined. In some embodiments, the analytical column comprises particles having an average diameter of about 5 μm. In some embodiments, the analytical column is a functionalized silica or polymer-silica hybrid, or a polymer particle or monolithic silica stationary phase (e.g., a phenyl-hexyl functionalized analytical column).

[0025] As used herein, an "ion-pairing reagent" is a salt, a chemical additive that electrostatically binds to the solute or analyte being chromatographed or subjected to other analytical processes according to the nominal chemical nature of the ion-pair complex, thereby allowing the manifestation of a particular chromatographic modality. Examples of ion-pairing reagents include alkylsulfonates and alkylammonium salts.

[0026] Analytical columns may be distinguished from typical 96-well format "extraction columns" or "extraction plates," which are typically used to separate or extract retained materials from unretained materials to obtain a "purified" sample from the matrix starting point to a significantly cleaner extracted endpoint for further purification or analysis.

[0027] Oligonucleotide therapeutics can be short DNA or RNA oligomers designed to interfere with the expression of disease-associated proteins, the sequence of which can be from about 15 nucleotide units to about 50 nucleotide units.

[0028] As used herein, the term "mass spectrometry" or "MS" generally refers to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio or "m / z." In MS techniques, one or more molecules of interest are ionized and then the ions are introduced into a mass analyzer where, due to a combination of electric fields, the ions follow paths in space that are dependent on their mass ("m") and charge ("z").

[0029] In certain embodiments, the mass spectrometer uses a "quadrupole" system. In a "quadrupole" mass spectrometer or "quadrupole ion trap" mass spectrometer, ions in an oscillating radio frequency (RF) electric field experience a force proportional to the direct current (DC) potential applied between electrodes, the amplitude of the RF signal, and the m / z. The voltage and amplitude can be selected such that only ions with a particular m / z travel the length of the quadrupole while all other ions are deflected. Thus, a quadrupole device can act as both a "mass filter" and a "mass detector" for ions injected into the device.

[0030] In certain embodiments, "tandem mass spectrometry" (MS / MS) is used. Tandem mass spectrometry (MS / MS) is the name given to a family of mass spectrometry techniques in which a "parent or precursor" ion generated from a sample is fragmented to produce one or more "fragment or product" ions, which are then mass analyzed by a second MS procedure. MS / MS techniques are useful for the analysis of complex mixtures, particularly biological samples, in part because the selectivity of MS / MS can minimize the need for extensive sample cleanup before analysis. In one example of an MS / MS technique, precursor ions are generated from a sample and passed through a first mass filter (quadrupole 1 or Q1) to select ions with a particular mass-to-charge ratio. These ions are then typically fragmented by collision with neutral gas molecules in the second quadrupole (Q2) to produce product (fragment) ions that are selected in the third quadrupole (Q3) and their mass spectra recorded by an electron multiplier detector. The product ion spectrum produced indicates the structure of its precursor ion, and the two mass filtering stages can remove ions from interfering species present in conventional mass spectra of complex mixtures.

[0031] As used herein, the terms "ionization" and "ionizing" refer to the process of producing analyte ions that have a net charge equal to one or more electron units. A negative ion is an ion that has a net negative charge of one or more electron units, while a positive ion is an ion that has a net positive charge of one or more electron units.

[0032] As used herein, the term "electron ionization" refers to a method in which an analyte of interest in the gaseous or vapor phase interacts with a stream of electrons. Collisions of those electrons with the analyte produce analyte ions that can then be subjected to mass spectrometry techniques. As used herein, the term "chemical ionization" refers to a method in which a reagent gas (e.g., ammonia) is subjected to electron impact, and analyte ions are formed by interaction of the reagent gas ions with the analyte molecules.

[0033] As used herein, the term "electrospray ionization" or "ESI" refers to a method in which a solution is passed through a short length of capillary tube and a high positive or negative potential is applied to the end of the tube. Upon reaching the end of the tube, the solution may be vaporized (atomized) into a plume or spray of very small droplets of solution in a solvent vapor. This mist of droplets may flow through an evaporation chamber where it is slightly heated to prevent condensation and evaporate the solvent. As the droplets become smaller, their surface charge density increases until such time that natural repulsion between like charges causes the release of ions as well as neutral molecules.

[0034] The term "atmospheric pressure chemical ionization" or "APCI" refers to a mass spectrometry method similar to ESI, but APCI generates ions through ion-molecule reactions that occur in a plasma at atmospheric pressure. The plasma is sustained by an electric discharge between the spray capillary and a counter electrode. The ions are then typically drawn into a mass analyzer by use of a set of differentially pumped skimmer stages. A counterflow of dry, preheated N2 gas may be used to improve solvent removal. Gas-phase ionization in APCI can be more effective than ESI for analyzing less polar species.

[0035] The term "atmospheric pressure photoionization" ("APPI") refers to a form of mass spectrometry in which the mechanism for photoionization of a molecule M is photon absorption and electron ejection to form M+ of that molecule. Because the photon energy is typically just above its ionization potential, the ion of that molecule is less likely to dissociate. In many cases, it may be possible to analyze samples without the need for chromatography, thus saving significant time and expense. In the presence of water vapor or protic solvents, the ion of the molecule may abstract H to form MH+. This tends to occur when M has a high proton affinity. This does not affect the accuracy of quantification, since the sum of M+ and MH+ is constant. Drug compounds in protic solvents are usually observed as MH+, while nonpolar compounds (e.g., naphthalene or testosterone) usually form M+.

[0036] (method) Disclosed herein is an embodiment of a method for oligonucleotide analysis using a novel solid-phase extraction and hydrophilic interaction liquid chromatography method.Due to the polar nature of some oligonucleotides, it can be extremely difficult to purify oligonucleotides by reversed-phase chromatography.Then, such polar entities can typically be accompanied by ion-pairing agents when purified by reversed-phase chromatography, so that polar oligonucleotides can have increased retention time and increase separation resolution.As a result, ion-pairing conditions have excessive equipment downtime caused, reduced column life, system dedication to ion-pairing method, high signal drift risk, and overall sensitivity loss in the dedication system.

[0037] Recently, efforts have been made to create a quantitative bioanalytical approach for oligonucleotides without the use of ion-pairing reagents, focusing in particular on hydrophilic interaction chromatography (HILIC) preceded by polymer-based weak cation exchange (WAX) solid phase extraction (SPE). Extraction and chromatography are two elements of such methods that are useful prior to mass spectrometry detection. In this context, a bioanalytical separation method for oligonucleotides has been developed, in which a novel and separate extraction step is shown to be the crux of this methodology. The focus of this methodology is on a unique retention scheme based on polarity to produce high recovery extractions to provide a high performance alternative extraction means, and further to include hydrophilic interaction liquid chromatography and modern high resolution MS as its endpoints. A method for the bioanalysis of oligonucleotides is disclosed herein that uses a novel hydrophilic solid phase extraction means with robust high recovery and a pair-free methodology, which combines all elements of the methodology to reach picomolar (pM) sensitivity.

[0038] Many embodiments of the present disclosure may include the general steps of performing a solid phase extraction, isolating the component of interest by chromatography, and performing mass spectrometry on the component of interest. In some embodiments, a method for determining the amount and / or presence of a compound of interest includes the steps of (i) providing a sample believed to contain the compound of interest; (ii) performing an extraction of the compound of interest, the extraction being a hydrophilic phase extraction; (iii) isolating the compound of interest from other components of the sample by liquid chromatography, and (iv) analyzing the compound of interest by mass spectrometry. In some embodiments, the sample is a biological sample, and the biological sample may be plasma. In some embodiments, the compound of interest may be an oligonucleotide or oligonucleotides, and may be an RNA oligonucleotide or a DNA oligonucleotide. In some embodiments, the oligonucleotide may be a therapeutic oligonucleotide. In some embodiments, the compound of interest may be an oligonucleotide therapeutic agent. In some embodiments, the method may be generally performed under ion-pair-free conditions. In some embodiments, the solid phase extraction may be a hydrophilic phase extraction performed on a modified sorbent bed comprising an aminopropyl phase on a silica base. In some embodiments, RNA oligonucleotides can be the compound of interest due to their high polarity.In some embodiments, DNA oligonucleotides can be the compound of interest.In some embodiments, RNA oligonucleotides can be more polar than DNA oligonucleotides, as indicated by the extra hydroxylation of their ribose rings.

[0039] In some embodiments, conditions similar to solid phase extraction (SPE) may be used during the extraction process. The sorbent bed for the extraction may vary but may include an aminopropyl phase on a silica base in a 96-well format. In one embodiment, the collection plate for the extraction is a polypropylene plate. In some embodiments, the extraction may be performed by gravity to allow elution to obtain a slow linear flow rate that is conducive to forming and breaking high energy ionic interactions depending on the biosample being analyzed. In some embodiments, the sorbent bed may be conditioned with an experimental mixture of acetonitrile and formic acid. In one embodiment, the sorbent bed is conditioned with a mixture of 9:1 (volume:volume) acetonitrile:2% formic acid (aqueous solution). In some embodiments, an equilibration step is not performed on the sorbent bed before loading the sample.

[0040] In some embodiments, the biological sample may be conditioned or diluted with various solvents before loading the biological sample onto the sorbent bed. In some embodiments, the biological sample may be plasma containing RNA oligonucleotides, and the sample is diluted with an internal standard and an aqueous acid. In one embodiment of the present disclosure, 100 μl of plasma is diluted with 700 μl of 6% aqueous phosphoric acid in 9:1 (volume:volume) acetonitrile:water, and 20 μl of 2500 nM internal standard. After the unique one-step conditioning of the sorbent bed with 1 mL of 9:1 (volume:volume) acetonitrile:(2% formic acid (aqueous)) and the biological sample preparation described above, the biological sample may be loaded onto the sorbent bed and then washed. In some embodiments, the wash may include a mixture of organic and aqueous liquids. In one embodiment, the sorbent bed is washed with 1 ml of 9:1 (volume:volume) acetonitrile:2% formic acid (aqueous). The sorbent bed containing the loaded sample is then washed with 1 ml of 8:2 (vol:vol) acetonitrile:2% ammonium hydroxide (aqueous). In other embodiments, alternative wash conditions may be used and more or fewer washes may be performed experimentally. Elution of the analyte may be performed by a final application of a polar wash solution. In one embodiment using RNA oligonucleotides as the compound of interest, the oligonucleotides may be eluted into a 96-well collection plate with 1 ml round wells by two successive applications of 400 μl of 2% ammonium hydroxide in 70:30 (vol:vol) water:acetonitrile. The eluate may be evaporated to dryness at 30° C.-45° C. using nitrogen gas and then resuspended for chromatography. In one example of the extraction process, the resuspension medium may include a high quality solvent mixture prior to injecting the biological sample into a chromatography system.

[0041] In some embodiments, the method of analyzing the oligonucleotide after the extraction step may include further isolating the compound of interest by chromatography. In some embodiments, the method for chromatographic separation may include liquid chromatography including an analytical column. The method may include experimentally adjusting the composition of the mobile phase. In some embodiments, the method may include experimentally adjusting the composition of the gradient to be used. In some embodiments, an isocratic mixture may be utilized after the extraction step. In one embodiment, the composition of the migration path may be a mixture of organic and aqueous liquids.

[0042] In some embodiments, the extracted and isolated compound of interest can be analyzed by a mass detection system. In some embodiments, the analyzing step can include ionizing the compound of interest and monitoring for parent peak ions and / or fragment ions to provide evidence of the compound of interest. In some embodiments, the analyzing step can include ionizing in negative ion mode and monitoring parent ions and fragment ions.

[0043] (system) A system for carrying out the method disclosed herein is also described.In some embodiments, the system for determining the amount and / or presence of a compound of interest in a sample from a subject can include a solid phase extraction system, a liquid chromatography system, and a mass spectrometry detection system.In some embodiments, the sample can be a biological sample.Furthermore, the biological sample can be plasma.In some embodiments, the compound of interest can be an oligonucleotide or a plurality of oligonucleotides.In some embodiments, the oligonucleotide can be a therapeutic oligonucleotide.

[0044] In some embodiments, the system comprising the solid phase extraction system can generally comprise a modified sorbent bed comprising an aminopropyl phase on a silica base, although the composition of the sorbent bed can vary.Therefore, the solid phase extraction system can be considered a hydrophilic phase extraction system.In many embodiments, the system comprising the solid phase extraction system, liquid chromatography system and mass spectrometry detection system can comprise and be used with a reagent that does not contain ion pairs.The mass spectrometry detection system can be a low-resolution mass spectrometer or a high-resolution mass spectrometer.

[0045] In one embodiment, the computer program product, when implemented on one or more data processors, may cause the one or more data processors to perform operations that direct at least one of the following steps: providing a sample suspected of containing a compound of interest, performing a solid phase extraction of the compound of interest, isolating the compound of interest from other components of the sample by liquid chromatography, and analyzing the compound of interest by mass spectrometry.

[0046] Some embodiments of the present disclosure may include analyzing the compound of interest by LC-MS after the step of hydrophilic phase extraction. In some embodiments, the LC-MS system may include a station for isolating the compound of interest from other compounds in the sample by chromatography, and a station for providing mass spectrometry by detection of nominal and accurate mass. In some embodiments, the compound of interest resuspended in a previously determined volume after the extraction step may be placed in an autosampler of a chromatography system. The autosampler may be configured to inject a certain amount of sample to be separated by an analytical column into the liquid chromatography system. In some embodiments, the autosampler may be configured to provide the system with different amounts of sample at different times by different injections. In one embodiment, the liquid chromatography system may be an ultra-performance liquid chromatography (UPLC) system. The UPLC system may include one or more pumps, a degasser, an autosampler, a column heater, and a mobile phase preheater.

[0047] In some embodiments, the liquid chromatography system may use an analytical column. In one embodiment, the analytical column is a regular-phase amide-based chromatography column with dimensions 2.1 mm×50 mm and 1.7 μm particle size. In some embodiments, the column may be an ethylene-bridged hybrid trifunctional bonded amide phase column, although other normal-phase columns of various compositions and dimensions may be used. An aqueous-organic gradient of increasing ratio may be used to aid in the isolation of the compound of interest. Similarly, in one embodiment, the mobile phase may include the components 0.05% ammonium hydroxide in 10 mM ammonium formate (aqueous) and acetonitrile. The gradient may be generated empirically. According to one embodiment, the gradient cycle may start with 20% aqueous and increase to 50% aqueous over 5 minutes. A 0.5 minute isocratic hold may be used during the last minute of the 6.5 minute period before the equilibration step. In other embodiments, a longer hold time may be used for more difficult separations. In other embodiments, the gradient may be altered to start with a composition less than 20% aqueous, while the final composition may be greater than 50% aqueous.

[0048] In some embodiments, various flow rates may be utilized according to the specifications of the liquid chromatography system. In one embodiment, an optimal flow rate of 0.45 ml / min was used for the mobile phase, with an injection of 6.0 μl. The amount injected into the system may correlate with the size of the injection loop, the size of the column, and the flow rate of the mobile phase. A wash may follow re-equilibration of the system. The composition of the mixture for the wash may be 0.05% ammonium hydroxide in 10 mM ammonium formate (aqueous solution) according to one embodiment. Alternatively, the composition of the wash solution may be 1:1 (volume:volume) acetonitrile:(0.05% ammonium hydroxide (aqueous solution) in 10 mM ammonium formate (aqueous solution)).

[0049] In some embodiments, after chromatographic separation, the liquid chromatography system may be configured to provide the mass spectrometer with a volume to be analyzed. In some embodiments, the mass spectrometer may analyze the compound of interest through chemical ionization followed by collision-induced fragmentation. In one embodiment, electrospray ionization (ESI) may be used by the system. In an alternative embodiment, atmospheric pressure chemical ionization (APCI) may be used by the system to ionize and fragment the compound of interest. Other ionization modes may be utilized by the system. In one embodiment, the mass spectrometer may be a nominal mass detector (low resolution mass spectrometer), while in another embodiment, the mass spectrometer may be an accurate mass detector (high resolution mass spectrometer). The nominal mass detector may include a triple quadrupole system. In an alternative embodiment, the accurate mass detector may include a time-of-flight tandem mass spectrometry (TOF-MS / MS) system. Both the nominal mass system and the accurate mass system may be operated in negative ion selection mode. In many embodiments, the system can be used with reagents that do not contain ion pairs.

[0050] FIG. 1 shows a flow chart for sample preparation and hydrophilic phase extraction according to an embodiment of the present disclosure. In FIG. 1, the sorbent bed is conditioned with a mixture of 9:1 (vol:vol) acetonitrile:2.0% formic acid (aq.). After dilution and preparation of the biological sample with aqueous acid and an internal standard, 800 μl of the biological sample is loaded onto the sorbent bed. The sorbent containing the sample containing the compound of interest is then washed with multiple washes, each wash varying in composition. Finally, the compound of interest can be eluted with multiple applications of 400 μl of 70:30:2 (vol:vol:vol) water:acetonitrile:(c.) ammonium hydroxide. After evaporation, the compound of interest is reconstituted with 200 μl of 4:6 (vol:vol) acetonitrile:(0.05% ammonium hydroxide in 10 mM ammonium formate (aq.).

[0051] FIG. 3 shows a three-dimensional bar graph showing the LC-MS response comparison of the analyte GNV705 AS and the internal standard according to an embodiment of the present disclosure, where an initial plasma volume of 100 μl was diluted to various volumes before loading. The bar graph is obtained using the LC-MS response of both the internal standard and the compound of interest GNV705 AS after hydrophilic phase extraction and separation. Such a comparison may be determined experimentally. Each increasing x-axis volume is recorded as the plasma volume after dilution with 6% aqueous acid containing the internal standard. The plasma was diluted to various volumes before loading onto the sorbent bed. The dilution with aqueous acid allows the basic groups on the oligonucleotide to be protonated so that an LC-MS response can be produced. Prior to the creation of FIG. 3, dilution tests showed approximately 25% better recovery when diluted with 4% to 6% phosphoric acid at the established conditions. The assay appeared to be optimized with a dilution of 700 μl of 6% phosphoric acid. In one embodiment of the present disclosure, more than 6% aqueous acid is used to dilute the plasma. Additionally, more than 700 μl of aqueous acid may be used to dilute the plasma.

[0052] FIG. 4 shows a bar graph of an elution volume optimization test according to an embodiment of the present disclosure. The volume optimization test can help determine the optimal eluent volume to use during extraction so that the compound of interest is not diluted beyond the detection threshold for mass spectrometry. Conversely, a minimum volume amount can be useful to provide sufficient sample volume to be injected and analyzed by the mass spectrometer. Thus, providing a challenge in determining the concentration to volume ratio. After washing the compound of interest on the sorbent bed during the extraction step, an elution step using 2.0% ammonium hydroxide in 3:7 (volume:volume) acetonitrile:water ensured that oligonucleotide GNV705 AS was completely solubilized and eluted into a 96-well collection plate. First, an elution volume of 200 μl provided insufficient volume to provide a useful response by mass spectrometry. It can be seen in FIG. 4 that an elution volume adjusted from 400 μl to 900 μl provided an optimal response. In one example, 800 μl of eluent (2.0% ammonium hydroxide in 3:7 (vol:vol) acetonitrile:water) was used to provide optimal results. In other embodiments of the present disclosure, volumes greater than 900 μl may be used to elute the compound of interest from the sorbent bed. Conversely, volumes less than 200 μl may also be used to elute the compound of interest from the sorbent bed.

[0053] Various embodiments of the present disclosure have been described herein. It should be recognized that these embodiments are merely illustrative of the present disclosure. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the above description. It is anticipated that those skilled in the art may use such variations as appropriate, and it is intended that the present disclosure be practiced otherwise than as specifically described herein. Accordingly, the present disclosure includes all modifications and equivalents of the subject matter described in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure unless otherwise indicated or otherwise clearly contradicted by context. EXAMPLES

[0054] (Example) Example 1 - HILIC and HPE method development (Chemicals and Materials) Reference material for analyte GNV705 AS (as well as N-1 and N-2 metabolite reference materials) was kindly gifted from Genevant Sciences (MA, USA). The method was to be quantitatively optimized only for single-stranded antisense, not double-stranded. Candidate internal standard (IS) reference materials, structural analogs, were obtained from Integrated DNA Technologies (IL, USA). Acetonitrile, concentrated phosphoric acid (85%), concentrated ammonium hydroxide (25%), concentrated formic acid and ammonium formate were all obtained from Sigma Aldrich and were LC-MS grade, except for phosphoric acid, which was ACS grade. Water was purified in-house using a ThermoScientific Barnstead Nanopure purification system by reverse osmosis filtration followed by deionization to a resistivity of 18.2 MΩ·cm. Control cynomolgus monkey plasma containing K2EDTA anticoagulant was obtained from BioIVT (NY, USA) and included sources from six individual donors for differential testing and selectivity.

[0055] (Calibration standards and quality control samples) GNV705 AS primary solutions and candidate IS were prepared at 100 μM in 9:1 (volume:volume) water:acetonitrile. Calibration and quality control (QC) sample spike solutions, as well as IS spike solutions, were prepared in 9:1 (volume:volume) water:acetonitrile in polypropylene tubes. Plasma was prepared in similar polypropylene tubes at concentrations of 0.5 nM, 2 nM, 5 nM, 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1000 nM, and 2000 nM for calibrator samples, and 0.5 nM, 2 nM, 5 nM, 10 nM, 20 nM, 100 nM, and 2000 nM for QC samples. Many of these nominal levels were the same between the calibrators and quality controls (QCs), and this was deemed to have no impact on the consistency of the results. The volume of the spike solution was 2% or less of the volume of the plasma to be spiked. Blank hemolytic plasma was prepared for spiking by adding previously flash frozen human whole blood to blank normal cynomolgus monkey plasma with K2EDTA from the main stock, resulting in a hemolysis level of 2%.

[0056] (Method development and performance evaluation) There were three major stages in this method development that were of most interest for some embodiments of the present invention: achieving linearity in the solution domain using appropriate HILIC-selected reaction monitoring (SRM) conditions, extraction from the matrix, and evaluation and comparison of detector formats.

[0057] The chromatography was easily established very similar to the previously reported work [MacNeill et al., Bioanalysis, 11:1155-67 (2019)], however, upon injection of the complete calibration curve in solution at representative concentrations for plasma extracts, it became clear that an unusual curvature appeared, whose slope increased with the nominal concentration. Related experiments to subsequently generate and confirm linearity were performed in both nominal and exact mass detection, which involved two important parameters. The first was to investigate the linearity or not from injection of solutions of various compositions of acetonitrile and (0.05% ammonium hydroxide in 10 mM ammonium formate [aqueous]). The second consisted in choosing how many easily detectable charge states to use in the quantification. The most abundant 4-charge state was available at m / z 1832, the 5-charge state at m / z 1465, and the 6-charge state at m / z 1221.

[0058] Towards the end of method development, focusing on the silica base as part of the foundation for a novel selectivity strategy for extraction, key SPE experiments were performed to investigate how elution volume related to extraction recovery and subsequently how the degree of plasma dilution prior to loading affected recovery. The former experiments used the following total elution volumes, each split into two applications: 200 μl, 400 μl, 600 μl, 800 μl and 900 μl. The latter experiments used the following addition volumes of 6% phosphoric acid (aq): 200 μl, 400 μl, 600 μl, 700 μl and 800 μl.

[0059] A major part of the final goal was to show the robust quantitative performance of the complete method developed for GNV705 AS, which was demonstrated using a series of analytical batches that mainly included accurate mass detection for interday and intraday analysis. There was a direct comparison of nominal and accurate mass detection for one intraday analysis for nominal mass detection, and one batch was later reinjected as one of the interday analyses for accurate mass. The method validation-style evaluation otherwise included basic experiments in the GLP validation domain, and establishment of recovery, selectivity, matrix factors, differential matrix effects, and determination of hemolytic effects were all properly performed. Stability was not directly investigated, but was previously characterized by other means.

[0060] (Establishing linearity) In the dissolution region, prior to the extraction study, tests using nominal mass detection included the most abundant precursor ions first, with the aim of summing the final total peak area based integrals for the most sensitive quantification. This premise is generally consistent with biopharmaceutical quantification using LC-MS. However, it was observed that a curve regression occurred, with the curvature of such properties of response increasing with nominal concentration. It was surprising that this could occur, especially since we knew from our experience with common oligonucleotide assays in our institution (data not shown) that this type of curvature is usually indicative of nonspecific adsorption phenomena to the container surface. This was based on previous oligonucleotide studies using the same type of polypropylene container and solvent composition, where the unpaired oligonucleotides, in all their unlimited polarity, have negligible affinity for the hydrophobic surface presented by the polypropylene.

[0061] Finally, it was demonstrated that adjusting the composition of the injected solvent to increase the solubility of the oligonucleotide therein by increasing its aqueous content was only a supporting part of the solution to the curvature. This could go from 45% aqueous to 60% aqueous without compromising the chromatography. The main factor was in the monitoring scheme within the acquisition method. Deviating from the usual expectations for biologics, it was found that including only the most abundant 4-charge state at m / z 1832, one precursor from the SRM, was a straight path to better linearity. Therefore, the other two mass spectrally visible charge states (5-charge state at m / z 1465 and 6-charge state at m / z 1221) were omitted. The various product ions listed for this one precursor could still be summed in the quantification. The product moment correlation coefficient for the various conditions is an indication of the improvement. With all charge states monitored in combination with only 45% aqueous in the injection composition, the coefficient was 0.836. Using the same aqueous composition and moving to just the precursor ion of that one significant charge state, the coefficient jumped to 0.980. With the final touch of moving to a 60% aqueous solution and including that one significant charge state, the correlation coefficient was 0.997.

[0062] These conditions were fully reproduced with respect to accurate mass detection. They were firmly held during conversion to matrix extract using 60% aqueous reconstitution. Furthermore, the curvature initially observed involving multiple precursor ions was also confirmed to be evident with accurate mass detection.

[0063] (Optimized sample preparation) SPE of GNV705 AS from cynomolgus monkey plasma was performed as follows. The SPE sorbent was NAX, 100 mg, from United Chemical Technology (PA, USA), an aminopropyl phase on a silica base in a 96-well format. The 96-well 1 ml round-bottom collection plate to receive the final eluent was regular DNA LoBind polypropylene from Eppendorf (Hamburg, Germany). Each step of applying liquid was passed through gravity alone. The time it took for this to occur was 3-4 min, corresponding to a linear flow rate slow enough to provide the best performance in an environment where high energy ionic interactions are formed and disrupted.

[0064] Candidate internal standard (IS) at 2500 nM in 1:9 (vol:vol) acetonitrile:water was added as a 20 μl aliquot to 100 μl plasma in 1.5 ml regular polypropylene tubes. This resulted in an IS concentration of 500 nM in the matrix. Each tube was then vortexed for 2 seconds. This was followed by the addition of 700 μl of 6% H3P04 (in water) to each sample and another vortex step.

[0065] Conditioning of the sorbent involved application of 1 ml of 9:1 (vol:vol) acetonitrile:2% formic acid (aq.) without a subsequent equilibration step prior to loading the diluted plasma sample. The prepared sample (800 μl) was loaded onto the conditioned sorbent bed, followed by application of a 1 ml wash containing 9:1 (vol:vol) acetonitrile:2% formic acid (aq.). The next wash was with 1 ml of 8:2 (vol:vol) acetonitrile:2% ammonium hydroxide (aq.). Elution of the analytes was performed by applying 400 μl of 70:30:2 (vol:vol:vol) water:acetonitrile:(c.) ammonium hydroxide twice to a 96-well collection plate with 1 ml round wells. The pH of the aqueous component of the mixture is 11.5. Finally, the eluates were evaporated under oxygen-free nitrogen at 40° C. and then reconstituted with 200 μl of 4:6 (vol:vol) acetonitrile:(0.05% ammonium hydroxide in 10 mM ammonium formate (aq.). The block was then sealed and placed on a plate shaker at 500 rpm for 10 minutes, then placed in the autosampler compartment at 10° C. to await injection.

[0066] Optimized LC-MS Analysis for Research-Based Method Development The analytical column for GNV705 AS quantification was a Waters (MA, USA) Acquity PREMIER UPLC BEH Amide with dimensions 2.1 mm x 50 mm and 1.7 μm particle size. The LC-MS front end was a typical Waters Acquity UPLC system equipped with a pump, degasser, autosampler, column heater and mobile phase preheater. The autosampler compartment was maintained at 10°C. Gradient elution was used with mobile phase components (0.05% ammonium hydroxide in 10 mM ammonium formate (aqueous)) and acetonitrile delivered at 0.45 ml / min, with both mobile and stationary phases at 40°C. For each gradient cycle, the mobile phase composition started at 20% aqueous and the mobile phase composition was linearly excursed to 50% aqueous over the next 5.0 min. This composition was maintained for 0.5 min, followed by re-equilibration for the remaining 1.0 min of the total run time of 6.5 min. The injection volume was 6.0 μl and partial loop mode with needle overfill was used with a strong wash composition of 0.05% ammonium hydroxide in 10 mM ammonium formate (aq), and a weak wash composition of 1:1 (vol:vol) acetonitrile:(0.05% ammonium hydroxide in 10 mM ammonium formate (aq).

[0067] (nominal mass detection) The triple quadrupole mass spectrometer was a Sciex (Concord, ON, Canada) 6500+ using Turbo Ionspray source conditions and the auxiliary gas was heated to 550° C. For the IonDrive source, the probe position was 0 mm vertically and 7 mm horizontally. The instrument was operated in high mass mode. There was no split of the LC flow to the ion source.

[0068] In negative ion SRM mode, the transitions used were 1832.4→586.1, 1832.4→664.1 and 1832.4→604.1 for GNV705 AS, where all values ​​indicate m / z. The precursor ion involved was [M-4H] 4- These peaks were selected based on the most plausible intensity and verifiability. Peak area based integration for quantitative endpoints included the sum of peaks acquired at each transition.

[0069] (Accurate mass detection) The high resolution mass spectrometer was a Sciex ZenoTOF 7600 System using an Optiflow ion source, the auxiliary gas was heated to 550°C, and there was no split of the LC flow into the source. Negative MRM using Zenotrap pulsing HR Acquisition was applied for quantification, the precursor ion m / z target was set as 1832.3, and the MS / MS scan range was 100 m / z to 2000 m / z. The simultaneously acquired TOF-MS data was analyzed by MRM analysis using Zcnotrap pulsing. HR It was quickly found that this gave a response nearly ten times lower than that obtained. Thus, for GNV705 AS, extracted ion chromatograms of the transitions 1832→586.1134, 1832→664.0810 and 1832→604.1199 (all values ​​indicate m / z) were generated using Sciex OS software with its extraction window width set to 0.02 Da. The precursor ions involved were the same as for the nominal mass, [M-4H] 4- The product ions corresponded to the charge state of 1670→586.1275, 1670→664.0609 and 1670→604.1013. The selected product ions were similar to the nominal mass scheme. These peaks were selected based on the most plausible intensity and verifiability. For the internal standard (IS), the extracted ion chromatograms were 1670→586.1275, 1670→664.0609 and 1670→604.1013 with the same extraction window width. The peak area based integration for the quantitative endpoint included the sum of the peaks in each transition.

[0070] (HILIC method development) The concentration of ammonium hydroxide in the aqueous component of the mobile phase was increased from 0.02% to 0.05%, and the gradient was developed over 5.0 min at a flow rate of 0.45 ml / min. These results in a much greater eluotropic force, which is necessary to provide timely elution of the RNA oligonucleotides. RNA oligonucleotides are more polar than DNA oligonucleotides, mainly due to the extra hydroxylation on their ribose units, and therefore are more strongly retained than RNA oligonucleotides.

[0071] Figure 2 shows a representative chromatogram, which illustrates the resolution at hand. This was a 5 μl injection of the 200 nM mixture in solution in the nominal mass detector. The N-1 and N-2 metabolites (which elute first and mostly co-elute with each other) are baseline separated from GNV705 AS, which elutes immediately after. The candidate internal standard, which elutes last, is then baseline separated from GNV705 AS.

[0072] (Development of hydrophilic phase extraction method) An initial attempt using a 100 mg bed mass of UCT NAX (aminopropyl bonded phase) obtained from United Chemical Technologies resulted in higher recoveries than previously observed. A series of tests have since allowed for complete optimization of the procedure.

[0073] For the 100 mg packed sorbent, the pre-elution procedure was adequate based on 1 ml of solvent application despite the sample load. A minimum of 10% aqueous was maintained in all steps to preserve HILIC conditions and to avoid oligonucleotide precipitation that may result in inaccuracies. In a rare and interesting discovery, we found that omitting the highly aqueous equilibration step proved beneficial for recovery in this process, as is intuitive in almost all SPE procedures. This hypothesizes that the artifacts of the retained oligonucleotide bands are so hydrophilic that they are repelled from the surrounding induction environment that would otherwise exist after the conditioning step, thereby not spreading as they would otherwise after the highly aqueous step, avoiding the associated recovery losses. Similarly, an initial fully aqueous wash step was applied to avoid breakthrough in the highly aqueous conditions that fully solubilize the nucleic acids (e.g., analytes).

[0074] Thus, the single condition was 1 ml of 9:1 (vol:vol) acetonitrile:2% formic acid (aq), whose acidity maintained the ionized state of the important ionic moieties of the bonded phase. The post-load wash was identical to the condition, again with its highly "oligo-phobic" organic nature helping to prevent analyte breakthrough. For dilution and loading of the plasma samples, it was decided to use 6% phosphoric acid (aq) as the diluent first. This was a bit more concentrated than that used in previous studies, and matched better with the low pH plateau achieved by phosphoric acid at increasing concentrations. This only seemed to make a difference for these first few moments, while high energy electrostatic bonds formed, involving a bit more neutralization of the phosphate backbone, and thus more non-polar interactions, which are low energy and therefore appear quickly. This is a phenomenon synonymous with the success of this condition in typical mixed-mode SPE. A quick test showed about 25% better recovery at 4%-6% phosphoric acid in separately established conditions. Figure 3 shows data from a study in which a plasma volume of 100 μl was diluted to various degrees before loading, a condition similar to that in at least one other customer-related application of oligonucleotides where a dependency in recovery was established in the sense that the recovery increased with the total volume applied (plasma and diluent) until a maximum value was reached (data not shown). We speculate that the reasoning has to do with maximizing the opportunity for capture by volumetrically spacing the species to be retained under a flow as slow as is practically reasonable. The reason is that, as mentioned above, it is the high energy electrostatic binding that needs to be manifested. Gravity alone provided a suitable flow for this study, and the filling of the wells allowed flows that amounted to a maximum of 4 minutes for each step. With regard to the results of the study, as shown in Figure 3, any such dependency did not seem to be overwhelming for GNV705 AS, but was more evident for its internal standard (IS), which showed a very definitive signal increase supported by triplicate analysis. The maximum value of 800 μl appeared to be optimal for safety and practicality.

[0075] As stated above, the first wash was identical to the single step conditions, 1 ml of 9:1 (vol:vol) acetonitrile:2% formic acid (aqueous). It was noted throughout that there was never any flow restriction through the sorbent. This indicated the absence of any protein precipitates, which largely confirmed their removal after denaturation in the sample dilution and inaccessibility to pores in the packed material that had slipped through in the sample load. The next wash just prior to elution was with 1 ml of 8:2 (vol:vol) acetonitrile:2% ammonium hydroxide (aqueous). This introduced a little eluotropicity by increasing the aqueous content and featuring a slightly alkaline pH without using a sufficient concentration to neutralize significant ionic moieties in the bonded phase, resulting in sustained retention. Elution was then applied with 2% ammonium hydroxide in (3:7 acetonitrile:water), which is highly aqueous enough to completely solubilize the analytes and alkaline enough to neutralize the sorbent. The final elution volume optimization results are shown in Figure 4. For the 100 mg packed bed, an excluded volume of 150 μl was expected, therefore a test with a total volume of 200 μl was included. As can be seen in FIG. 4, this was not enough to result in complete elution. Once 2-3 excluded volumes had passed, a full 400 μl was the full response corresponding to the elution of what remained in the cartridge observed. To err on the side of caution, with the knowledge of how incident samples can differ quite significantly in their natural matrix composition, the final elution volume was established at a total of 800 μl to provide sufficient elution power in unusual conditions that may call for it. Notably, as can be seen in FIG. 4, the area between 600 μl and 900 μl showed no difference in recovery index.

[0076] Crucially, in all available numerical details for low QC (LQC) and high QC (HQC) nominal levels {20 nM and 2000 nM), the recovery for GNV705 AS is above 60% and its candidate internal standard (IS) in the same region calculated for the replicate set at its relevant 1 nominal level. The final HPE schematic is shown in Figure 1. The name "hydrophilic phase extraction" (HPE) was assigned to this new scheme, since the root of its retention mechanism is embedded in polar interactions similar to HILIC.

[0077] On the surface, it may be acknowledged that the coupling of HILIC-based sample extraction with a HILIC LC-MS analytical endpoint may not result in a clear orthogonality of selectivity between these two methodological components. However, this feature may in fact be largely conserved due to the details of each case. Primarily, the chemistry of the stationary support differs between the analytical column and the solid-phase packed cartridge, amide on hybrid polymer-silica versus aminopropyl on silica. The extraction then uses a comprehensive washing scheme including both acidic and alkaline applications prior to elution, which has a major bearing on selectivity and on the elimination or resolution of interferences.

[0078] Example 2 - Example of a Specific Embodiment Non-limiting examples of specific embodiments of the present technology are listed herein.

[0079] A1 1. A method for determining the amount and / or presence of a compound of interest in a sample from a subject, comprising: providing a sample suspected of containing said compound of interest; performing an extraction of said compound of interest, said extraction being a hydrophilic phase extraction; isolating said compound of interest from other components of said sample by liquid chromatography; and analyzing the compound of interest by mass spectrometry A method comprising:

[0080] A2 The method of embodiment A1, wherein the sample is a biological sample.

[0081] A3 The method of embodiment A2, wherein the biological sample is plasma.

[0082] A4 The method of embodiment A3, wherein said compound of interest is an RNA oligonucleotide or a DNA oligonucleotide.

[0083] A5 The method of embodiment A4, wherein said RNA or DNA oligonucleotide is a therapeutic oligonucleotide.

[0084] A6 The method of embodiment A1, wherein the hydrophilic phase extraction is carried out on a sorbent bed comprising an aminopropyl phase on a silica base.

[0085] A7 The step of performing hydrophilic phase extraction comprises: adding an aqueous acid and an internal standard to said sample; conditioning the sorbent bed with a first volume of 9:1 (vol:vol) acetonitrile:2.0% formic acid; loading the sample onto the sorbent bed; washing the sorbent bed with a second volume of 9:1 (v:v) acetonitrile:2.0% aqueous formic acid; washing the adsorbent bed with a third volume of 8:2 (vol:vol) acetonitrile:2.0% ammonium hydroxide; and Elute the compound of interest into a collection plate by applying two successive volumes of 2% ammonium hydroxide in 70:30 (vol:vol) water:acetonitrile. The method of embodiment A6, comprising:

[0086] A8 isolating the compound of interest, using an ethylene bridged hybrid amide phase column to isolate said compound of interest; Using an aqueous-organic mobile phase containing both 0.05% ammonium hydroxide in 10 mM ammonium formate and acetonitrile; and Use a gradient ranging from 20% aqueous to 50% aqueous over 5 minutes. The method of embodiment A1, comprising:

[0087] A9 The method of embodiment A1, wherein the step of analyzing the compound of interest comprises ionizing the compound of interest with the mass spectrometer and monitoring fragment ions in negative ion mode.

[0088] A10 The method of embodiment A1, wherein the step of analyzing the compound of interest by mass spectrometry is performed by both a low resolution mass spectrometer and a high resolution mass spectrometer.

[0089] A11 The method of embodiment A1, performed under ion-pair-free conditions.

[0090] B1 1. A system for determining the amount and / or presence of a compound of interest in a sample from a subject, comprising: Solid phase extraction systems; A liquid chromatography system; and Mass Spectrometer System Including, the system.

[0091] B2 The system of embodiment B1, wherein the sample is a biological sample.

[0092] B3 The system of embodiment B1, wherein the biological sample is plasma.

[0093] B4 The system of embodiment B1, wherein said compound of interest is an RNA oligonucleotide or a DNA oligonucleotide.

[0094] B5 The system of embodiment B4, wherein the RNA or DNA oligonucleotide is a therapeutic oligonucleotide.

[0095] B6 The system of embodiment B1, wherein the solid phase extraction system is a hydrophilic phase extraction system, the hydrophilic phase extraction system comprising a modified sorbent bed comprising an aminopropyl phase on a silica base.

[0096] B7 The system of embodiment B1, comprising a reagent that does not include an ion pair.

[0097] B8 The system of embodiment B7, wherein the mass spectrometer system is a low resolution mass spectrometer or a high resolution mass spectrometer.

[0098] C1 When implemented in one or more data processors: providing a sample containing a compound of interest; performing a solid phase extraction of said compound of interest from said sample; isolating the compound of interest from other components of the sample by liquid chromatography; and Analyzing the compound of interest by mass spectrometry. 5. A computer program product for causing one or more data processors to perform operations that direct at least one of:

Claims

1. 1. A method for determining the amount and / or presence of a compound of interest in a sample from a subject, comprising: providing a sample suspected of containing said compound of interest, said sample being a biological sample, and said compound of interest being an RNA oligonucleotide or a DNA oligonucleotide; performing an extraction of the compound of interest, the extraction being a hydrophilic phase extraction carried out on a sorbent bed comprising an aminopropyl phase on a silica base, (a) adding an aqueous acid and an internal standard to the sample; (b) conditioning the sorbent bed with a solution containing 9:1 (volume:volume) acetonitrile:2.0% formic acid; (c) loading the sample onto the sorbent bed; (d) washing the adsorbent bed; and (e) eluting the target compound with a solution containing ammonium hydroxide and acetonitrile; the steps of: isolating the compound of interest from other components of the sample by liquid chromatography; and analyzing the compound of interest by mass spectrometry A method comprising:

2. The method of claim 1 , wherein the biological sample is plasma.

3. The method of claim 1 , wherein the RNA or DNA oligonucleotide is a therapeutic oligonucleotide.

4. isolating said compound of interest using an ethylene-bridged hybrid amide phase column to isolate the compound of interest; Using an aqueous-organic mobile phase containing both 0.05% ammonium hydroxide in 10 mM ammonium formate and acetonitrile; and Use a gradient ranging from 20% aqueous to 50% aqueous over 5 minutes The method of claim 1 , comprising:

5. 10. The method of claim 1, wherein the step of analyzing the compound of interest comprises ionizing the compound of interest with the mass spectrometer and monitoring fragment ions in negative ion mode.

6. 10. The method of claim 1, wherein the step of analyzing the compound of interest by the mass spectrometer is performed by both a low-resolution mass spectrometer and a high-resolution mass spectrometer.

7. The method of claim 1 carried out under ion-pair-free conditions.

8. 1. A system for determining the amount and / or presence of a compound of interest in a sample from a subject, comprising: an adsorbent bed comprising an aminopropyl phase on a silica base, and (a) adding an aqueous acid and an internal standard to the sample, wherein the sample is a biological sample; (b) conditioning the sorbent bed with a solution containing 9:1 (volume:volume) acetonitrile:2.0% formic acid; (c) loading the sample onto the sorbent bed; (d) washing the adsorbent bed; and (e) eluting the target compound with a solution containing ammonium hydroxide and acetonitrile; wherein the compound of interest is an RNA oligonucleotide or a DNA oligonucleotide; a liquid chromatography system; and Mass Spectrometer System Including, the system.

9. The system of claim 8 , wherein the biological sample is plasma.

10. The system of claim 8 , wherein the RNA or DNA oligonucleotide is a therapeutic oligonucleotide.

11. The system of claim 8 , comprising a reagent that does not contain an ion pair.

12. The system of claim 11 , wherein the mass spectrometer system is a low resolution mass spectrometer or a high resolution mass spectrometer.

13. When implemented on one or more data processors: providing a sample containing a compound of interest, wherein the sample is a biological sample and the compound of interest is an RNA oligonucleotide or a DNA oligonucleotide; performing a solid phase extraction of the compound of interest from the sample, the extraction being a hydrophilic phase extraction carried out on a sorbent bed comprising an aminopropyl phase on a silica base, the step of performing the hydrophilic phase extraction comprising: (a) adding an aqueous acid and an internal standard to the sample; (b) conditioning the sorbent bed with a solution containing 9:1 (volume:volume) acetonitrile:2.0% formic acid; (c) loading the sample onto the sorbent bed; (d) washing the adsorbent bed; and (e) Eluting with a solution containing ammonium hydroxide and acetonitrile the steps of: isolating the compound of interest from other components of the sample by liquid chromatography; and analyzing the compound of interest by mass spectrometry a tangible, non-transitory computer-readable medium storing a computer program product that causes one or more data processors to perform operations that direct at least one of:

14. The method of claim 1, wherein (d) comprises a first washing step comprising washing the adsorbent bed with a solution comprising acetonitrile and formic acid.

15. The method of claim 14, wherein the first washing step comprises washing the adsorbent bed with a solution containing 9:1 (volume:volume) acetonitrile:2.0% formic acid.

16. The method of claim 14, wherein (d) further comprises a second washing step comprising washing the adsorbent bed with a solution comprising acetonitrile and ammonium hydroxide.

17. The method of claim 16, wherein the second washing step comprises washing the adsorbent bed with a solution containing 8:2 (volume:volume) acetonitrile:2.0% ammonium hydroxide.

18. The method of claim 1, wherein the eluting comprises eluting the target compound into a collection plate by applying two successive volumes of the solution comprising ammonium hydroxide and acetonitrile.

19. The method of claim 1, wherein the eluting comprises eluting the target compound into a collection plate by applying two successive volumes of 2% ammonium hydroxide in 70:30 (volume:volume) water:acetonitrile.

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