Two-dimensional LC-MS / MS system

Abstract

This invention provides a novel 2D LC analysis method for analyzing one or more analytes in a source sample. [Solution] The LC column of the first liquid chromatography system is equipped with a flow divider valve and is directly connected to the second LC column. The flow divider valve is set to a first position for a first predetermined time so that the solvent effluent from the first LC column is directed to the wastewater. The flow divider valve is set to a second position for a second predetermined time so that the solvent effluent from the first LC column enters the second LC column for further separation. The sample separated on the chromatograph obtained from the second LC column is analyzed by a mass spectrometer.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority from U.S. Provisional Application 62 / 855,636, filed on 31 May 2019, the disclosures of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) has emerged as a powerful technique for detecting and measuring a wide variety of analytes. It has rapidly become the preferred method for accurately and precisely quantifying analytes from biological matrices. A major obstacle in LC-MS / MS analysis of biological matrices is the presence of major matrix components such as phospholipids, proteins, or nucleotides, which interfere with the identification and quantification of the target analyte present in these matrices (see, for example, Non-Patent Documents 1 and 2). As a fundamental step in the development of bioanalytical methods, sample washing is required to remove major matrix contaminants before sample analysis. Methods used for sample washing are broadly classified into either "offline," referring to hands-on sample preparation procedures, or "online," referring to sample preparation procedures performed in a liquid chromatography (LC) system (see Non-Patent Documents 3, 4, and 5).

[0003] One of the most common offline sample purification methods is solid-phase extraction (SPE), which focuses on removing matrix components and concentrating the analyte of interest using a sample preparation cartridge (see also Non-Patent Literature 6, and Non-Patent Literature 3, 4, and 5 mentioned above). The SPE extract containing the analyte of interest is dried and reconstituted in a different solvent system, which must be suitable for one-dimensional (1D) LC-MS / MS analysis. The process of drying and reconstituting the sample in a different solvent system is called "solvent exchange" or "buffer exchange." This process is necessary because the solvent systems used for SPE and those used for one-dimensional LC-MS / MS analysis are not compatible. The solvent exchange process leads to sample loss and reduced reproducibility. Furthermore, SPE cartridges are expensive and the process is time-consuming. To eliminate the SPE step for sample purification, online two-dimensional (2D) LC-MS / MS was developed. 2D LC-MS / MS uses two column systems. The first column is often made of hydrophilic resin (normal-phase chromatography) and is used to remove major matrix contaminants before introducing the sample of interest into the second column. The second column can be made of hydrophobic resin (reverse-phase chromatography) and further separates the analyte of interest from other interfering molecules before introducing it into the mass spectrometer (Non-Patent Documents 7, 8, 9, and 10). As a result, conventional online 2D LC-MS / MS analytical methods employ two different solvent systems. This requires a dedicated pump system for each column, as well as multi-port diversion valves to allow for analyte transfer and solvent exchange. These requirements make conventional 2D LC-MS / MS methods very complex and expensive, outweighing the potential advantages of 2D LC-MS / MS. Most laboratories are not equipped to perform this type of assay.

[0004] Thus, there is a need for improved analytical methods that are both precise and convenient. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Carmical and Brown, Biomed.Chromatogr.A(2016)30:710-20 [Non-Patent Document 2] Massood et al., Lipids(2012)47:209-26 [Non-Patent Document 3] Afonso-Olivares et al.(2017)1487:54-63 [Non-Patent Document 4] Mokh et al., Sci Total Environ.(2017)609:830-41 [Non-Patent Document 5] Dong et al., Bioanalysis(2015)7:2227-33 [Non-Patent Document 6] Vanol et al., Biomed.Chromatogr.(2017)32:1-10 [Non-Patent Document 7] Iguiniz and Heinisch, J Pharmaceut Biomed Anal.(2017)145:482-503 [Non-Patent Document 8] Ling et al., Biomed.Chromatogr.(2014)28:1284-93 [Non-Patent Document 9] Pirok et al., Anal Chem.(2019)91:240-63 [Non-Patent Document 10] Iguiniz et al., Talanta.(2019)195:272-80 [Summary of the Invention] [Means for Solving the Problems]

[0006] This disclosure provides a novel 2D LC analysis method for analyzing one or more analytes in a source sample. The method includes (a) extracting one or more analytes from a source sample with an extraction solvent to obtain an extract sample; (b) applying the extract sample and solvent system to a first liquid chromatography (LC) column equipped with an LC system, wherein the first LC column is directly connected to a second LC column via a tube equipped with a flow divider valve; (c) setting the flow divider valve to a first position at a first predetermined time so that the solvent effluent from the first LC column is directed to a wastewater; (d) setting the flow divider valve to a second position at a second predetermined time so that the solvent effluent from the first LC column enters the second LC column for further separation; (e) repeating steps (c) and (d) as necessary; and (f) analyzing the separated sample on a chromatograph obtained from the second LC column. An LC system refers to an LC instrument equipped with a pump system for introducing the solvent system into the LC column. "Direct" means that there is no second LC system dedicated to the second column, and therefore the method utilizes only one LC system for both dimensions. In some embodiments, step (f) includes analyzing the chromatographically separated sample obtained from the second LC column using mass spectrometry (MS), such as tandem MS.

[0007] In some embodiments, LC is high-performance liquid chromatography or ultrahigh-performance liquid chromatography. In some embodiments, the first LC column is a normal-phase column and the second LC column is a reversed-phase column, or vice versa.

[0008] In some embodiments, the solvent system for 2D LC includes one or more of methanol, acetonitrile, and water. In further embodiments, the solvent system further includes ammonium acetate and / or formic acid.

[0009] "Solvent system" means a solvent mixture or combination used during an LC run to analyze a target analyte. During a run, the solvent composition can change (e.g., by varying the relative ratios of the components of the solvent mixture), but such changes do not lead to the need to perform solvent exchange on the sample as it moves from the first column to the second column. In some embodiments, the relative ratios of the components of the solvent system change during the sample run. In some embodiments, the relative ratios of the components of the solvent system change during the sample run.

[0010] The source sample may be a biological sample such as a tissue sample, serum, plasma, blood, dried blood spot, urine, saliva, sputum, tears, cerebrospinal fluid, semen, or feces. In some embodiments, one or more analytes are proteins, lipids, carbohydrates, nucleotides, metabolites, vitamins, hormones, or steroids.

[0011] In certain embodiments, one or more analytes are ceramide and lyso-sphingomyelin, and the source sample is derived from the blood of a patient with acid sphingomyelinase deficiency. In further embodiments, ceramide and lyso-sphingomyelin are extracted from the blood sample using an extraction solvent containing 80% methanol (v / v), 15 - 20% acetonitrile (v / v), 0 - 5% water (v / v), 10 mM ammonium acetate, and 1% formic acid. In certain embodiments, the first LC column is a silica column and the second LC column is a C18 column (i.e., the resin of the column is made of a polymer with 18 carbon atoms). In further embodiments, the solvent system contains 0.5% trifluoroacetic acid.

[0012] In some embodiments, the solvent systems applied to the first and second LC columns include 0 to 85% methanol (v / v), 0 to 15% acetonitrile (v / v), and 0 to 100% water (v / v). In further embodiments, the solvent system is made by mixing a first solvent containing water and 0.5% trifluoroacetic acid with a second solvent containing 85% methanol (v / v), 15% acetonitrile (v / v) and 0.5% trifluoroacetic acid. In certain embodiments, the ratio of the first solvent to the second solvent is 70:30, 85:15, or 99:1.

[0013] Other features, objects, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description is illustrative and not restrictive, showing embodiments and aspects of the present invention for purposes of example only. Various changes and modifications within the scope of the present invention will be apparent to those skilled in the art from the detailed description.

Brief Description of the Drawings

[0014] [Figure 1] It is a schematic diagram showing the configuration of a conventional online 2D LC-MS / MS. [Figure 2] It is a diagram comparing the configurations of offline 1D LC-MS / MS, conventional online 2D LC-MS / MS, and the present online continuous flow 2D LC-MS / MS (single LC system). [Figure 3] It is a schematic diagram showing the continuous flow 2D LC-MS / MS methodology of the present invention. [Figure 4] It is a diagram showing the analysis of ceramide and lyso-sphingomyelin (lyso-SPM) using the continuous flow 2D LC-MS / MS methodology of the present invention. In this methodology, ceramide and lyso-SPM were separated from contaminated phospholipids.

Modes for Carrying Out the Invention

[0015] This disclosure provides a novel method for simultaneously analyzing multiple analytes in a source sample using continuous flow two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS / MS). In the 2D LC-MS / MS method of the present invention, a single solvent system is used as the extraction solvent as well as the mobile phases in both dimensions of the 2D LC-MS / MS analysis. Between the 2D LC portions of the analysis run, a flow-diversion valve located between the two columns is switched at intervals to various positions so that the solvent flow from the first column flows to either the waste liquid or the second column for further separation. Thus, in this method, two or more liquid chromatographs are analyzed. While a roughy (LC) column can be used, only one LC pump system (i.e., LC system) is required because the need for solvent exchange is avoided. Furthermore, the solvent flow in this method is continuous and is not interrupted by a solvent exchange step or a second LC system. In contrast, conventional 2D LC-MS / MS uses two different solvent systems, for example, one polar and one nonpolar, for two separation mechanisms. As a result, two LC systems are required.

[0016] One major advantage of this method is the elimination of a second pump system, which simplifies the subsequent 2D LC-MS / MS configuration (e.g., by using a flow-splitting valve with fewer ports). The elimination of the second pump system significantly reduces instrument cost and method deployment time. This innovation allows 2D LC-MS / MS analysis to be performed in any laboratory currently using standard 1D LC-MS / MS analysis. This innovation provides flexibility to analytical laboratories, allowing them to choose between 1D or 2D LC-MS / MS analysis using the same instrument. The system simplification allows for faster personnel training, reduces the likelihood of assay failures, and shortens sample handling time. Furthermore, this method is suitable for multiplexing, significantly reducing sample preparation and analysis time, as well as sampling bias, and improving reproducibility. In addition, the novel 2D-LC techniques described herein can be easily adapted to operations using non-mass spectrometer detectors, such as charged aerosol detectors (CADs), light scattering detectors, and UV detectors. This flexibility significantly increases the applicability of this analytical method.

[0017] These improvements will enable this analytical method to be widely used in the fields of biochemical analysis for biopharmaceutical research, medical diagnostics, and environmental surveys, utilizing 2D LC-MS / MS technology.

[0018] Sample preparation This method can be used to analyze (e.g., detect and / or quantify) one or more analytes of interest in any sample matrix, such as biological or environmental samples. Biological samples may be samples from humans, plants, animals, or any biological organ, such as cell and tissue cultures, tissue biopsies, whole blood, dried blood spots, plasma, deproteinized plasma, serum, deproteinized serum, semen, sputum, urine, feces, sweat, saliva, bile, tears, cerebrospinal fluid, swabs from body parts, skin, and hair. Environmental samples may be air samples, soil samples, water samples, food samples, and any substance samples. Analytes of interest may be small molecules such as active pharmaceutical ingredients, as well as biomolecules such as polypeptides, peptides, nucleic acids, lipids or fatty acids, carbohydrates, hormones, vitamins, steroids, and metabolites.

[0019] Before applying a sample to a 2D LC system, the analyte of interest can be concentrated and separated by filtration, precipitation, centrifugation, extraction, dilution, or a combination thereof, to remove most contaminants and interfering substances. For example, solid-phase extraction (SPE) can be used to concentrate the desired component from a source sample. In SPE, the analyte of interest is concentrated using a sample preparation cartridge. The SPE extract containing the analyte may be dried and reconstituted in a solvent system compatible with the 2D LC system. If the SPE extraction solvent is compatible with the 2D LC system, as in some embodiments of this method, the drying and reconstitution steps are not necessary.

[0020] The target analyte can also be extracted from the source sample by liquid-liquid extraction (LLE). LLE is used to separate analytes based on their relative solubility in two immiscible or partially miscible liquids (typically a polar solvent such as water and a non-polar organic solvent). The target analyte is first partitioned by the solvent, and then extracted, concentrated, and diluted.

[0021] The target analyte can also be extracted from the source sample by solid-supported liquid-liquid extraction (SLE). In SLE, an aqueous solution of the sample is loaded onto a support made of diatomaceous earth. After the sample is adsorbed onto the support, it is washed several times with an organic extraction solvent such as methyl tert-butyl ether. After the target analyte is separated into the organic phase, it is concentrated by drying and then reconstituted in a solvent suitable for a 2D LC system, such as 50:50 methanol:water.

[0022] If the target analyte is a protein, it may be concentrated from the source sample by protein precipitation extraction (PPE). Protein precipitation methods may include desalting, isoelectric focusing precipitation, and organic solvent extraction. As an example, the source sample is prepared for 2D LC loading by desalting. This protein precipitation method is based on the fact that proteins "salt out" from the solution as the concentration of a neutral salt such as ammonium sulfate increases. In another example, the source sample is prepared by isoelectric focusing precipitation. This method may be used to precipitate contaminating proteins rather than the target protein. The isoelectric point (pI) is the pH at which the net primary charge of a protein is zero. For most proteins, the pI is in the pH range of 4 to 6. Inorganic acids such as hydrochloric acid and sulfuric acid may be used as precipitants. A potential drawback of isoelectric focusing precipitation is the irreversible denaturation caused by inorganic acids.

[0023] The solvent used to extract and concentrate the target analyte from a source sample may be compatible with a 2D LC system. That is, the extraction solvent containing the extracted analyte may be loaded directly into the 2D LC system without the need for solvent exchange. In some embodiments, the extraction solvent for biomolecules (e.g., polypeptides, peptides, nucleic acids, lipids, hormones, vitamins, steroids, and carbohydrates) comprises methanol, acetonitrile, and / or water, and the ratio of these three substances can be varied depending on the target analyte. For example, to extract lipids from a blood sample or tissue, the solvent contains a mixture of methanol, acetonitrile, and water in 100% total volume percentages, e.g., about 30-100% methanol (v / v), about 0-100% acetonitrile (v / v), and about 0-50% water (v / v). The solvent may optionally contain other components, e.g., 10 mM ammonium acetate and 1% formic acid. For example, the extraction solvent may consist of 80% methanol, 15% acetonitrile, 5% water, 10 mM ammonium acetate, and 1% formic acid, or 80% methanol, 20% acetonitrile, 10 mM ammonium acetate, and 1% formic acid. See also Chuang et al., Methods Mol Biol. (2016) 1378:263-72. The specific solvent composition will depend on the properties of the target analyte and the interference matrix.

[0024] Two-dimensional liquid chromatography For example, a source sample can be processed by concentrating the target analyte, and then the processed sample can be fed into a liquid chromatography pump system for application to a first liquid chromatography column.

[0025] Liquid chromatography (LC) is a process in which a fluid solution (mobile phase) passes through a column of substances (stationary phase) that have been finely divided by capillary action, selectively retaining one or more components of the fluid solution. The selective retention of components in the fluid solution by the stationary phase is due to the higher affinity of the components to the stationary phase than to the mobile phase. Liquid chromatography as used herein includes high-performance liquid chromatography (HPLC), ultrahigh-performance liquid chromatography (UHPLC), highly turbulent liquid chromatography (HTLC), normal-phase chromatography (NPC), reverse-phase chromatography (RPC), supercritical fluid chromatography (SFC), affinity chromatography, ion exchange chromatography (IEX), capillary liquid chromatography, electrochromatography, membrane chromatography, monolithic chromatography, nano- and capillary liquid chromatography. This includes, but is not limited to, chromatographic chromatography and size exclusion chromatography (SEC). The analyte of interest may be retained in the stationary phase and then eluted, or it may flow through the stationary phase without being retained. The analyte in the eluate or effluent can be monitored by various means (e.g., UV, fluorescence, light scattering, or electrical conductivity) based on retention time, peak intensity, and peak area. Further detailed analysis of the analyte may be performed using techniques such as mass spectrometry, as described below.

[0026] A liquid chromatography (LC) system typically includes some or all of the following components: (i) Injector: Also known as a sample manager or autosampler, an injector is used to introduce a source sample into the mobile phase that is transferred to an LC column. (ii) Reservoir: The solvent reservoir holds the solvent system (mobile phase) used for separation in liquid chromatography. (iii) Pumps: Typically, high-pressure pumps are used to generate and maintain a predetermined flow rate of the mobile phase. Traditionally, each LC column required its own dedicated pump system. (iv) Columns: LC columns include an inlet port for receiving a sample and an outlet port for releasing effluent, and are typically packed with a solid adsorption medium such as silica, polymers, and other resins. LC columns may be, for example, normal-phase columns (usually hydrophilic), reverse-phase columns (usually hydrophobic), cation exchange columns, anion exchange columns, size exclusion chromatography columns, membrane columns, monolithic columns, nano or capillary LC columns, and chiral chromatography columns. (v) Valves: A switching valve is a high-pressure valve between the column and the next destination of the eluent or effluent passing through the column. The next destination may be, for example, a waste liquid collector or an analyte detector. Diverter (or divert) valves can be controlled manually or by computer and may include a trap column. Typically, diverter valves used in LC systems can have as many as 12 ports. (vi) Accessories: High-pressure tubing and fittings are used to connect the sample injector, solvent reservoir, pump, column, and detector and to form conduits for the mobile phase.

[0027] The 2D LC system has two LC columns with two orthogonal separation mechanisms. The first LC column is the first dimension ( 1 D) is called the second LC, and the second LC is the second dimension ( 2 This method is called D). A sample containing one or more target analytes is injected into a first LC column with a suitable solvent. The solvent passes through the column under high pressure, and the target analytes are separated from contaminants in the sample. The effluent from the first LC column is collected and injected into a second LC column, where the target analytes are further separated. Alternatively, 1 After retaining the D eluate in the trap column, it can be injected into the second dimension. 2 A guard column containing a stationary phase similar to that of the D column may be used as a trap column. After elution from the second dimension, the effluent containing the target analyte can be further analyzed.

[0028] There are generally two types of 2D LC. In comprehensive 2D LC (LC×LC), 1 the entire flow of the D effluent is 2 directed towards the D column. In heart-cut 2D LC (LC-LC), a specific effluent peak or a specific portion of the chromatogram is 2 directed towards the D column. Also, 2 multiple peaks or multiple portions of the chromatogram can be selected to be transferred to the D column. The split valve of the LC system can isolate and save multiple cuts, which are then 2 analyzed on the D column.

[0029] Figure 1 shows a typical heart-cut 2D LC system, with the solid lines indicating the flow direction of the mobile phase. In step 1, a first LC system with a normal-phase (NP) column separates the target lipid analyte from interfering phospholipids. A 10-port split valve directs the effluent containing the analyte towards the trap column, retaining the analyte with the trap column while the phospholipids exit the trap column and are sent to the waste collector. In step 2, a fluid compatible with a second LC column (reverse phase or RP) is input by a second LC pump system. This fluid flows through the trap column by controlling the position of the 10-port valve, achieving solvent exchange in the trap column. In step 3, a solvent of the second mobile phase is input by the second LC pump, passes through the trap column, and brings the analyte to the RP column by setting the position of the split valve. The analyte is further separated on the RP column and finally analyzed by a mass spectrometer.

[0030] The novel 2D LC system of this disclosure requires only one LC pump system. The flow from the first column to the second column can be continuous without requiring a solvent exchange step. This is possible because a single mobile phase solvent system is used. The composition of the mobile phase solvent system (e.g., relative ratio of components) can be adjusted over time as the analyte passes through the separation system, but because the solvent system will fit both columns even with its adjusted components, there is no need for solvent exchange when the analyte moves from one column to the next. A flow divider valve between the two columns can direct the effluent from the first column to waste or to the second column, depending on the expected effluent discharge time from the first column. Because there is no need for solvent exchange, the flow divider valve between the two columns can be made simpler, and fewer ports are required. The novel 2D LC system of this disclosure encompasses both comprehensive 2D LC and heartcut 2D LC systems.

[0031] Figure 3 shows a heart-cut embodiment of the novel 2D LC system of this disclosure. In step 1, the LC system introduces a sample into an NP column using a solvent system having a first mobile phase ratio, which then transports the specific analytes of interest to an RP column via the position of a 3-port diversion valve, where they are retained. In step 2 (i.e., a later point in time), the diversion valve is switched to a second position, directing the NP column effluent, which carries interfering matrix, to wastewater. In step 3, additional analytes from the NP column are transferred to the RP column, and all target analytes retained in the RP column are eluted by solvent systems of different mobile phase ratios introduced by the LC pump system. The target analytes eluted from the RP column are then subjected to further analysis, such as mass spectrometry. Because the solvent system throughout the entire LC run contains components of the same compatibility (although the ratio changes as the run progresses), there is no need to interrupt the solvent flow to perform solvent exchange. Furthermore, in contrast to conventional 2D LC systems, only one pump system (indicated as "LC system 1") is required.

[0032] In the novel system of the present invention 1D column and 2 The D column may be selected based on the properties of the target analyte and matrix interference components. In some embodiments, as illustrated above, 1 Column D is a normal-phase column. 2 Column D is a reversed-phase column. In some embodiments (for example, with respect to carbohydrate analytes), 1 Column D is a normal-phase column. 2 The D column is a weak anion exchange column. In some embodiments (for example, with respect to protein / peptide analytes), 1 Column D is a normal-phase column. 2 Column D is a reverse-phase column. In some embodiments (for example, with respect to oligonucleotide analytes such as circulating tumor cell (CTC) DNA), 1 Column D is a normal-phase column. 2 Column D is a reversed-phase column containing an ion pairing reagent.

[0033] LC solvents include water, methanol, ethanol, acetonitrile, trifluoroacetic acid, heptafluorobutyric acid, ether, hexane, ethyl acetate, and organic solvents, such as carbonized Examples of solvents include, but are not limited to, hydrogen solvents (e.g., aliphatic and aromatic solvents), oxygenation solvents (e.g., alcohols, ketones, aldehydes, glycol ethers, esters, and glycol ether esters), and halogenation solvents (e.g., chlorinated and brominated hydrocarbons). The LC solvent may be buffered and may contain ammonium acetate, ammonium formate, ammonium bicarbonate, acetic acid, trifluoroacetic acid, formic acid, trimethylamine, and triethylamine. In some embodiments, the solvent system used in this 2D LC system may contain methanol, acetonitrile, and water, which are compatible with the extraction solvent. In certain embodiments, the solvent system contains about 0-100% methanol, about 0-100% acetonitrile, and about 0-90% water, with a total volume percentage of 100%. In certain embodiments, the solvent system of the 2D LC system is a mixture of mobile phase A (mobile phase solvent A) and mobile phase B (mobile phase solvent B) in various ratios, where mobile phase A contains water and 0.5% trifluoroacetic acid, and mobile phase B contains 85% methanol, 15% acetonitrile, and approximately 0.5% trifluoroacetic acid.

[0034] As an example, a source sample containing one or more lipid analytes (e.g., sphingolipids, cholesterol, and triglycerides) is extracted with an extraction solvent containing 80% methanol, 15–20% acetonitrile, and 0–5% water (e.g., 80% methanol, 15% acetonitrile, and 5% water; or 80% methanol and 20% acetonitrile). The extraction solvent may optionally contain 10 mM ammonium acetate and 1% formic acid. The extracted sample is loaded into an LC system using a mobile phase solvent system containing acetonitrile, methanol, and water, and the lipid analytes are separated from major matrix contaminants (carbohydrates, proteins, nucleotides, etc.). The solvent system may further contain 10 mM ammonium acetate, 1% formic acid, and 0.5% trifluoroacetic acid. The first LC column is an NP column, in which lower polarity lipids elute first and move toward the reverse-phase column via a flow-splitting valve, while higher polarity lipids are retained in the NP column. Next, the flow diversion valve is switched to the waste position to remove the NP column effluent carrying matrix contaminants. Then, the flow diversion valve is returned to its original position, moving the higher polarity lipids already retained in the NP column to the second dimension for further separation. At the time of switching the valve, the solvent flow is adjusted to either the waste or the second column. In the RP column, higher polarity lipid analytes are eluted first, while lower polarity lipid analytes are retained in the column until a highly hydrophobic elution solvent is used. The separated lipid analytes can be further analyzed by methods such as mass spectrometry.

[0035] As an example, a sample containing one or more protein / peptide analytes, e.g., insulin, is extracted with an extraction solvent containing methanol, acetonitrile, and water. The extraction solvent may further contain 0.5% acetic acid and 0.01% trifluoroacetic acid. The extracted sample is then injected into a first column, e.g., a normal-phase column, using a mobile phase solvent system containing acetonitrile, methanol, and water to separate the protein / peptide analytes from major matrix contaminants (carbohydrates, lipids, nucleotides, etc.). The solvent system may further contain 0.5% acetic acid and 0.01% trifluoroacetic acid. In some embodiments, the first dimension is a normal-phase column, where more hydrophobic phospholipids are eluted first and more hydrophilic polypeptides are retained in the normal-phase column. In some other embodiments, the first dimension is an anion-exchange column, where positively charged polypeptides are eluted first and transferred to the second dimension via a flow-dividing valve, while negatively charged polypeptides are retained in the anion-exchange column. The flow-dividing valve is then switched to waste to remove matrix contaminants. The flow divider valve is returned to its original position to transfer the polypeptides already retained in the first dimension to the second dimension for further separation. The timing of valve switching to adjust the solvent flow to either the waste liquid or the second column, and the design of the solvent gradient, can be easily adjusted to separate different protein analytes and to eliminate matrix interference. In a further embodiment, the second dimension is a reversed-phase column, and polar proteins While the polar proteins are eluted first, the nonpolar proteins are retained in the column until a more hydrophobic elution solvent is used. The separated protein analytes can then be further analyzed, for example, by mass spectrometry.

[0036] As another example, a sample containing one or more nucleic acid analytes, such as synthetic oligonucleotides, is extracted with an extraction solvent containing water and acetonitrile. The extracted sample is then injected into a silica column, such as a normal-phase column, using a mobile phase solvent system containing methanol, acetonitrile, and water, to separate the nucleic acid analytes from major matrix contaminants (carbohydrates, lipids, proteins, etc.). The solvent system may further contain trimethylamine or triethylammonium bicarbonate. Next, the flow diversion valve is switched to waste liquid to remove matrix contaminants. The flow diversion valve is switched back to its original position to transfer the nucleic acid analytes already held in the first dimension to the second dimension for further separation. The timing of the valve switching to adjust the solvent flow to either waste liquid or the second column, and the design of the solvent gradient, can be easily adjusted to separate different nucleic acid analytes and to eliminate matrix interference. In a further embodiment, the second dimension is a C18 column using an ion pairing mechanism, such as a reversed-phase column. The separated nucleic acid analytes can then be further analyzed, for example, by mass spectrometry.

[0037] As an example, a sample containing one or more carbohydrate analytes, such as N-linked fetine oligosaccharides, is extracted with an extraction solvent containing methanol, acetonitrile, and water. The extraction solvent may further contain 10 mM ammonium acetate, 0.5% acetic acid, and 0.1% formic acid. The extracted sample is then injected into a silica column, such as a normal-phase column, using a mobile phase solvent system containing methanol, acetonitrile, and water to separate the carbohydrate analytes from major matrix contaminants (lipids, proteins, nucleic acids, etc.). The solvent system may further contain 10 mM ammonium acetate, 1% formic acid, and 0.5% trifluoroacetic acid. Next, the flow divider valve is switched to waste to remove matrix contaminants. The flow divider valve is returned to its original position, and the carbohydrates already held in the first dimension are moved to the second dimension for further separation. The timing of the valve switching to adjust the solvent flow to either the waste or the second column, and the design of the solvent gradient, can be easily adjusted not only to separate different carbohydrate analytes but also to eliminate matrix interference. In further embodiments, the second dimension is a weak anion exchange column. The decomposed carbohydrate analytes can then be further analyzed, for example, by mass spectrometry.

[0038] As an example, a sample containing one or more steroid analytes, e.g., dihydrotestosterone, is extracted with an extraction solvent containing methanol, acetonitrile, and water. The extraction solvent may further contain 10 mM ammonium acetate, 0.5% acetic acid, and 1% formic acid. The extracted sample is injected into a silica column, e.g., a normal-phase column, using a mobile phase solvent system containing methanol, acetonitrile, and water, to separate the steroid analytes from major matrix contaminants (carbohydrates, lipids, proteins, nucleic acids, etc.). The solvent system may further contain 10 mM ammonium acetate, 0.5% acetic acid, and 0.5% trifluoroacetic acid. Next, the flow divider valve is switched to waste to remove matrix contaminants. The flow divider valve is returned to its original position, moving the steroid analytes, already held in the first dimension, to the second dimension for further separation. The timing of the valve switching to adjust the solvent flow to either the waste or the second column, and the design of the solvent gradient, can be easily adjusted to separate different steroid analytes and to eliminate matrix interference. In further embodiments, the second dimension is a C18 column, for example, a reversed-phase column. The separated steroid analytes can be further analyzed by methods such as mass spectrometry.

[0039] mass spectrometry Analytes separated by the novel 2D LC system of the present invention can be further analyzed by optimal techniques. Often, mass spectrometry (MS) is used due to its ultra-high sensitivity. A mass spectrometer ionizes the target analyte, separates the ions based on their mass-to-charge ratio in a vacuum, and finally measures the intensity of each ion. Mass spectra are highly effective for qualitative and quantitative analysis because they can indicate the concentration levels of ions with specific masses.

[0040] A mass spectrometer consists of three main components: an ion source for ionizing the analyte, a mass spectrometer that separates ions based on the mass-to-charge ratio (m / z), and a detector that detects the separated ions. In an ionization device, 2The D effluent is sprayed, desolvated, and ionized to generate charged particles, or ions. The ions pass through a series of mass spectrometers under vacuum. Precursor ions of a specific (m / z) ratio are selected, and all particles of other (m / z) ratios are removed and passed through the mass spectrometer. The separated ions are then detected, for example, by an electron multiplier tube. Ionization of the sample may be performed, for example, by electrospray ionization (ESI), atmospheric pressure chemical ionization (ACPI), photoionization, electron impact ionization, chemical ionization, fast atomic impact (FAB) / liquid secondary ion mass spectrometry (LSIMS), matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray / plasma spray ionization, or particle beam ionization. The ions may be detected, for example, by multiple reaction monitoring (MRM), selective ion monitoring mode (SIM), selective reaction monitoring (SRM), or an electron multiplier tube.

[0041] MS data is sent to a computer, where voltage versus time is plotted. The concentration of one or more target analytes in the source sample can be determined by comparing the area under the peaks in the chromatogram to a calibration curve, or by comparing the ratio of the internal standard to the test sample.

[0042] In some embodiments of this disclosure, the mass spectrometry method utilizes "tandem mass spectrometry" or "MS / MS," in which the selected precursor ions are further fragmented into product ions by collision with an inert gas such as argon, helium, or nitrogen. A second mass spectrometer is used to target specific product ion fragments for detection. This selection-fragmentation-detection procedure can be further extended to the first generation of product ions. For example, the selected product ions can be further fragmented to generate a group of other product ions, and so on. The fragmentation patterns of the ions are highly specific to the structure of the compound, thus enabling precise structural determination.

[0043] In some embodiments, the mass spectrometer may be selected from quadrupole spectrometers, ion trap spectrometers, Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, electrostatic trap spectrometers, magnetic sector spectrometers, quadrupole ion trap spectrometers, and time-of-flight spectrometers (both MALDI and SELDI).

[0044] The 2D LC-MS system of this disclosure is used to obtain purified target analytes. 2 The interface further includes a relay interface from the D column to the mass spectrometer. This interface is used to address the inherent incompatibility between liquid chromatography and mass spectrometry. While the mobile phase solvent system in an LC system is a pressurized fluid, MS instruments generally operate under vacuum. This interface transfers the target analyte from the LC unit to the MS unit, removing most of the mobile phase solvent system used in the liquid chromatography separation process while preserving the chemical identity of the target analyte.

[0045] In some embodiments, the interface is an electrospray interface. Alternatively, the interface may be an atmospheric pressure ionization interface, an atmospheric pressure chemical ionization interface, a thermospray interface, or a mobile belt interface. This may be a face, a direct liquid introduction interface, a particle beam interface, or a fast atomic impact (FAB) based interface.

[0046] 2D LC-MS combines the superior separation capabilities of liquid chromatography with the excellent sensitivity and selective mass spectrometry capabilities of mass spectrometry to provide molecular mass and structural information of components in a mixture. This information can be supplemented by information obtained from other LC detectors, including but not limited to refractive index detectors, chiral detectors, radioflow detectors, UV detectors, fluorescence detectors, light scattering detectors, and electrical conductivity detectors.

[0047] application Novel 2D LC-MS / MS systems, including the novel 2D LC-MS / MS system described herein, can be used to analyze (including detection and quantification) a variety of molecules, including small molecules (e.g., active pharmaceutical ingredients) and large molecules (e.g., biomolecules).

[0048] In some embodiments, the 2D LC-MS / MS system of the present invention is used to monitor biomarkers in preclinical and clinical research as well as in the development of screening / diagnostic and therapeutic purposes. For example, the 2D LC-MS / MS system may be used to measure levels of specific lipid biomarkers in lysosomal storage diseases such as Fabry disease, Gaucher disease, Krabbe disease, and acid sphingomyelinase deficiency (ASMD; e.g., Niemann-Pick disease (NPD) types A, B, and A / B).

[0049] As further illustrated in the following examples, two lipid biomarkers, ceramide (CER) and lyso-sphingomyelin (lyso-SPM), can be analyzed in blood samples from ASMD patients using a 2D LC-MS / MS system. ASMD is a sphingolipid metabolic disorder characterized by the accumulation of sphingomyelin in tissues throughout the body, particularly the spleen, liver, lungs, bone marrow, and sometimes the brain. Elevated levels of lyso-SPM (sphingosine phosphocholine) are equally common. The enzyme deficient in patients, acid sphingomyelinase, catalyzes the hydrolysis of sphingomyelin in lysosomes, producing phosphocholine and ceramide. Because lipids such as ceramide and lyso-SPM are highly elevated in ASMD patients, they can be used as biomarkers for screening and diagnosing ASMD, and as monitors for enzyme replacement therapy (using recombinant human ASM such as olipudase alfa).

[0050] To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way. [Examples]

[0051] Example 1: 2D LC-MS / MS detection of ceramide and lyso-SPM in human plasma This example describes the use of the novel 2D LC-MS / MS system of this disclosure for the analysis of ceramide (CER) and lyso-SPM in human plasma samples.

[0052] Chemicals and reagents Standard reference ceramide (derived from porcine brain) was purchased from Avanti Polar Lipids (AL, USA). The internal CER standard (N-nonadecanoyl-D-erythro-sphingosine) and lyso-sphingomyelin standard (sphingosylphosphorylcholine) were purchased from Matreya (PA, USA). The internal lyso-SPM standard (d9-lysosphingomyelin) was synthesized in-house. Methanol and acetonitrile (both HPLC grade) were purchased from Honeywell (NC, USA). Ammonium acetate... Monium was purchased from Sigma Aldrich (MO, USA). Deionized water was obtained using the company's MilliQ DI system (Millipore, MA, USA). Formic acid (Optima® LC / MS grade) was purchased from Fisher Scientific (Hampton, NH). Trifluoroacetic acid (TFA) was purchased from EMD Millipore (MA, USA).

[0053] lipid extraction Ten μL of human plasma was mixed with 800 μL of assay workwater (80% methanol, 20% acetonitrile, 10 mM ammonium acetate, and 1% formic acid) containing an internal standard (IS) of CER (0.22 μg / mL) in a 96-well plate. The mixture was vortexed at 11,000 rpm for 10 minutes using a DVX-2500 multi-tube vortexer (VWR Scientific Products, NJ, USA), sonicated in a water bath (Branson 3510 Ultrasonic Cleaner, Marshall Scientific, NH, USA) for 10 minutes, and then centrifuged in a counter-centrifuge (Beckman Coulter, CA, USA) for 5 minutes. The supernatant was transferred to a new 96-well plate and subjected to 2D LC-MS / MS analysis. CER concentration in each sample was quantified as μg / mL, and lyso-SPM was quantified as ng / mL in plasma based on a calibration curve using linear fit.

[0054] LC-MS / MS System Configuration 2D LC-MS / MS analysis was performed using an Acquity UPLC system (Waters Corp., Milford, MA, USA) and an API 4000 quadrupole mass spectrometer (AB Sciex, Toronto, Canada). The 2D LC-MS / MS system was controlled by Analyst® software (AB Sciex). Data processing and analysis were performed using Microsoft Excel and Watson LIMS® software (Thermo Fisher Scientific, Waltham, MA).

[0055] The 2D LC configuration used a silica column (Ultra Silica column, 2.1 × 150 mm, 5.0 μm, Restek, PA, USA) for the first dimension and a C18 column (Acquity UPLC BEH C18 column, 2.1 × 100 mm, 1.7 μm, Waters Corp.) for the second dimension. Both HPLC columns were placed in the Acquity Column Manager so that each column occupied a different compartment within the Column Manager, and both columns were maintained at the same temperature (60°C). The 2D HPLC configuration included the following connections: a) A PEEK tube (approximately 60 cm in length) connecting the outlet of the one-dimensional analysis column to port 1 of the flow divider valve. b) A second PEEK tube (approximately 70 cm in length) connecting port 2 of the flow divider valve to the inlet of the two-dimensional analysis column. c) A third PEEK tube (approximately 80 cm in length) connecting the outlet of the two-dimensional analysis column to the mass spectrometer. d) A fourth PEEK tube (at least 60 cm in length) to connect port 3 of the flow divider valve to the waste liquid.

[0056] mobile phase Mobile phase A was water with 0.5% trifluoroacetic acid added. Mobile phase B was 85% methanol, 15% acetonitrile, and 0.5% trifluoroacetic acid. Mobile phases A and B were mixed in different ratios and loaded into the LC system at predetermined points in the run, as shown in Table 1 below. The injection volume was 2 μL relative to the sample, and the mobile phase flow rate was 0.2 mL / min.

[0057] [Table 1]

[0058] LC flow diversion valve conditions In conjunction with the solvent system preparation shown in the table above, the flow splitting valve of the 2D LC system is switched to a different port position at the predetermined time points shown in Table 2 below. 1Different fractions of the D effluent could be directed to either the waste liquid or the next (RP) column. See also Figure 4.

[0059] [Table 2]

[0060] mass spectrometry Multiple reaction monitoring (MRM) was used for the analysis of ceramide and lyso-SPM. This method uses a triple quadrupole MS to first target the ions corresponding to the target compounds, ceramide and lyso-SPM, and then fragment these target ions to generate various daughter ions. One (or more) of these fragmented daughter ions are selected for quantification. Only compounds that satisfy both of these criteria—that is, the specific parent ion and specific daughter ions corresponding to the compound's mass—are separated within the mass spectrometer.

[0061] The MRM channel parameters for the API 4000 mass spectrometer are shown in Table 3 below.

[0062] [Table 3]

[0063] Table 4 shows the ceramide internal standard (IS), lyso-SPM analytes, and MRM channel parameters of the lyso-SPM IS.

[0064] [Table 4]

[0065] result In this novel analytical method, CER and lyso-SPM were simultaneously extracted using a 2D LC system with an assay solution compatible with the mobile phase solvent system to be used later. The extracted sample was then directly injected into the first column and transferred to the second column via a continuous flow without the need for solvent exchange. Normal-phase chromatography was found to be suitable for separating CER and lyso-SPM from phospholipids. Therefore, this method was used as the first dimension of LC separation. A second dimension of separation using reversed-phase chromatography was employed to separate CER and lyso-SPM from structurally related compounds such as sphingoglycolipids and lyso-sphingoglycolipids.

[0066] More specifically, in this LC process, the flow divider valve was first set to position A to allow column flushing. Then the valve was set to position B, and the ceramide was separated from the phospholipids in the normal phase (NP) column and flowed into the RP column, where it was retained. Subsequently, the flow divider valve was switched to waste (position A), 1 The phospholipids and other matrix components following the D effluent were removed. Finally, the flow divider valve was switched back to position B to allow lyso-SPM to enter the RP column, separating both ceramide and lyso-SPM from other potential interfering molecules before mass spectrometry.

[0067] The data indicate that both CER and lyso-SPM were successfully extracted and quantified. As shown in Figure 4, ceramide eluted first from the NP column, followed by the major contaminant phospholipids, and then lyso-SPM. By setting the diversion valve to position A, phospholipids were directed towards the wastewater, and by setting the valve to position B, ceramide and lyso-SPM analytes were directed to the RP column. On the RP column, lyso-SPM appeared as a single peak, while the ceramide analyte was fractionated into distinct retention windows depending on the fatty acid chain length and the number of double bonds, including the following isoforms: C16, C18, C20 and C22:1, C22 and C24:1, C24 and C26:1. This data indicates that the CER isoforms eluted from the RP column as sharp peaks with low background noise, and a single peak was identified for each isoform. Several runs were performed, and consistent retention times were obtained.

[0068] The 2D LC-MS / MS method described above reduced the sample preparation time for quantifying CER and lyso-SPM in human plasma from a typical 8 days to just 2 days. Furthermore, the original labor-intensive offline extraction process, including SPE and protein precipitation (PPT) procedures for sample purification, was reduced from 25 hours to 1 hour by utilizing the extraction solvent and continuous-flow 2D LC of the present invention. See, for example, Chuang et al., Methods in Molecular Biology, Vol. 1378, DOI 10.1007 / 978-1-4939-3182-8_28. As seen above, the entire 2D LC run was completed in 20 minutes.

[0069] [Table 5]

[0070] Example 2: Assay Qualification for 2D LC-MS / MS Detection of Ceramide in Human Plasma This example describes a study qualifying a 2D LC-MS / MS system for detecting ceramide. In this study, samples, calibration standards, and controls were added to a 96-well plate. Next, assay work solution containing a known amount of internal standard (CER-IS) was added to the wells. Then, as described in Example 1, the plate was vortexed, The samples were sonicated and centrifuged. After centrifugation, the supernatant was transferred to a new 96-well plate. This new plate was then transferred to a 2D LC-MS / MS system for analysis, as described in Example 1.

[0071] After the sample run, the peaks corresponding to each individual CER isoform were integrated using Analyst® software. Next, the peak areas of each individual CER isoform for any given sample were summed in Excel. The total CER peak areas were imported into Watson®, and the total CER concentration (μg / mL) was calculated based on the ratio of the total CER peak area to the CER-IS peak area using the intercept and slope of the calibration curve.

[0072] Novel multiplex assays were qualified for CER quantification. Qualification parameters included system suitability, intra-run and inter-run precision and accuracy, linearity of calibration (standard) curves, analytical sensitivity, carryover, delipidated plasma matrix effect, reproducibility of reinjection, batch size evaluation, extraction recovery, sample dilution integrity, determination of CER criteria in healthy donors, and comparison of methods for determining plasma CER levels.

[0073] Carryover was assessed by injecting the solvent (assay-used solution only) after the highest calibration standard for each run. The delipidated plasma matrix effect was assessed using three independent lots of delipidated plasma spiked with three different concentrations of analytes (low quality control (LQC), medium quality control (MQC), and high quality control (HQC)). Reproducibility of reinjection was assessed by continuously reinjecting samples at LLOQ (lower limit of quantification), LQC, MQC, HQC, and ULOQ (upper limit of quantification) concentrations. Recovery of extraction was assessed by comparing two pooled patient samples before and after the addition of CER. An internal standard (IS) was introduced at the same stage with respect to pre- and post-addition conditions.

[0074] This data demonstrates that the novel 2D LC-MS / MS method of the present invention detected 11 CER isoforms using multiple reaction monitoring (MRM) with independent MS / MS channels for each of these isoforms. System suitability testing showed a %CV between 0% and 5% in all pass runs performed throughout the validation process. The average %Bias and average %CV (coefficient of variation) for all concentrations met established standards for precision and accuracy. Analytical sensitivity was defined as the lowest concentration of analyte that could be measured with acceptable precision and accuracy. The %CV and average %bias for LLOQ were 8% and -8%, respectively.

[0075] The minimum and maximum carryover of the analyte peak area were 2% and 11%, respectively. The overall %CV of the delipidated plasma matrix effect measured at three concentrations was 6% (LQC), 2% (MQC), and 3% (HQC). The %CV of reproducibility of reinjection ranged from 2% to 4% at specific concentrations. Extract recovery rates ranged from 67% to 88%, with %CV varying from 2% to 11%. The results demonstrate that this method is precise and accurate and suitable for use in measuring ceramide concentrations in human plasma.

Claims

1. A method for analyzing one or more analytes in a source sample, comprising the following steps: (a) A step of obtaining an extracted sample by extracting one or more analytes from a source sample with an extraction solvent, (b) A step of applying an extracted sample and a solvent system to a first liquid chromatography (LC) column equipped with a liquid chromatography (LC) system, wherein the first LC column is directly connected to a second LC column via a tube equipped with a flow divider valve, (c) A step of setting a flow divider valve to a first position at a first predetermined time so that the solvent effluent from the first LC column flows towards the waste liquid, (d) Setting a flow divider valve to a second position at a second predetermined time so that the solvent effluent from the first LC column enters the second LC column for further separation, (e) A process that repeats steps (c) and (d) as necessary, (f) A step of analyzing the sample separated by chromatography obtained from the second LC column. Methods that include...

2. The method according to claim 1, wherein step (f) comprises analyzing the chromatographically separated sample obtained from a second LC column using mass spectrometry (MS).

3. The method according to claim 2, wherein the MS is a tandem MS.

4. The method according to any one of claims 1 to 3, wherein LC is high-performance liquid chromatography or ultrahigh-performance liquid chromatography.

5. The method according to any one of claims 1 to 4, wherein the first LC column is a normal-phase column and the second LC column is a reversed-phase column, or vice versa.

6. The method according to any one of claims 1 to 5, wherein the solvent system comprises methanol, acetonitrile, and water, one or more of these.

7. The method according to claim 6, wherein the solvent system further comprises ammonium acetate and / or formic acid.

8. The method according to any one of claims 1 to 7, wherein the relative ratio of the components of the solvent system changes during a single sample run.

9. The method according to any one of claims 1 to 8, wherein the source sample is a biological sample.

10. The method according to claim 9, wherein the biological sample is a tissue sample, serum, plasma, blood, dried blood spot, urine, saliva, sputum, tears, cerebrospinal fluid, semen, or feces.

11. The method according to claim 9 or 10, wherein one or more analytes are proteins, lipids, carbohydrates, nucleotides, metabolites, vitamins, hormones, or steroids.

12. The method according to claim 11, wherein one or more analytes are ceramide and lyso-sphingomyelin, and the source sample is derived from the blood of a patient with acid sphingomyelinase deficiency.

13. The method according to claim 12, wherein ceramide and lyso-sphingomyelin are extracted from a blood sample using an extraction solvent comprising 80% methanol (v / v), 15-20% acetonitrile (v / v), 0-5% water (v / v), 10 mM ammonium acetate, and 1% formic acid.

14. The method according to claim 12 or 13, wherein the first LC column is a silica column and the second LC column is a C18 column.

15. The method according to claim 14, wherein the solvent system comprises 0.5% trifluoroacetic acid.

16. The method according to claim 14 or 15, wherein the solvent system applied to the first and second LC columns comprises 0-85% methanol (v / v), 0-15% acetonitrile (v / v), and 0-100% water (v / v).

17. The method according to claim 16, wherein the solvent system is obtained by mixing a first solvent comprising water and 0.5% trifluoroacetic acid with a second solvent comprising 85% methanol (v / v), 15% acetonitrile (v / v), and 0.5% trifluoroacetic acid.

18. The method according to claim 17, wherein the ratio of the first solvent to the second solvent is 70:30, 85:15, or 99:1.

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