Two-dimensional LC-MS / MS system

Abstract

To provide a novel method for detecting one or more analytes in a source sample by continuous flow 2D LC-MS / MS using a single LC system.SOLUTION: In step 1, a system inputs a sample obtained by solvent extraction to a normal-phase column, which is the first column, in the system. An outlet of the normal phase column is connected to a three-port diverting valve. An analyte of interest is input to a reverse-phase column, which is the second column. In step 2, the diverting valve is switched such that reverse-phase column effluent carrying interfering matrix is directed to waste. In step 3, additional analytes from the normal-phase column travel to the reverse-phase column, and all the target analytes retained by the reverse-phase column are eluted and subject to mass spectrometry analysis.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Application No. 62 / 855,636, filed May 31, 2019, the disclosure of which is incorporated herein by reference in its 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 is rapidly becoming the method of choice for accurate and precise quantification of 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 analytes of interest present in these matrices (see, for example, Non-Patent Documents 1 and 2). As a fundamental step in bioanalytical method development, sample cleanup is required to remove major matrix contaminants prior to sample analysis. Methods used for sample cleanup can be broadly categorized as either "offline," referring to a hands-on sample preparation procedure, or "online," referring to a sample preparation procedure performed on a liquid chromatography (LC) system (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 analytes of interest using sample preparation cartridges (see Non-Patent Document 6; see also Non-Patent Document 3, Non-Patent Document 4, and Non-Patent Document 5, cited above). The SPE extract containing the analytes of interest is dried and reconstituted in a different solvent system, which must be compatible with 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 system used for SPE is incompatible with that used for one-dimensional LC-MS / MS analysis. 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 a two-column system. The first column, often made of a hydrophilic resin (normal-phase chromatography), is used to remove major matrix contaminants before introducing the sample of interest into the second column. The second column can be made of a hydrophobic resin (reverse-phase chromatography) and further separates the analyte of interest from other interfering molecules before introduction into the mass spectrometer (Non-Patent Document 7, Non-Patent Document 8, Non-Patent Document 9, and Non-Patent Document 10). As a result, traditional online 2D LC-MS / MS analytical methods use two different solvent systems. This requires dedicated pumping systems for each column and multi-port diversion valves to allow analyte transfer and solvent exchange. These requirements make traditional 2D LC-MS / MS methods very complex and expensive, outweighing the potential benefits 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 simple. [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] [[ID=****]]

[0006] The present disclosure provides a novel 2D LC analytical method for analyzing one or more analytes in a source sample. The method includes: (a) extracting one or more analytes from the source sample with an extraction solvent to obtain an extracted sample; (b) applying the extracted sample and solvent system to a first liquid chromatography (LC) column equipped with an LC system, where the first LC column is directly connected to a second LC column via tubing equipped with a diversion valve; (c) setting the diversion valve to a first position at a first predetermined time so that the solvent effluent from the first LC column is directed to waste; (d) setting the diversion 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 chromatographically separated sample obtained from the second LC column. An LC system refers to an LC instrument equipped with a pump system for injecting the solvent system into the LC column. By "directly" is meant that there is no second LC system dedicated to the second column, and thus the method utilizes only one LC system for both dimensions. In some embodiments, step (f) comprises analyzing the chromatographically separated sample obtained from the second LC column using mass spectrometry (MS), such as tandem MS.

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

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

[0009] "Solvent system" refers to a solvent mixture or combination used during an LC run to analyze a target analyte. During the run, the solvent composition may be changed (e.g., by varying the relative ratios of the components of the solvent mixture). The relative ratios of the components of the solvent system may be changed (by changing the solvent content) but the change does not result in the need to perform a 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 are changed 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 stool, etc. In some embodiments, the one or more analytes are proteins, lipids, carbohydrates, nucleotides, metabolites, vitamins, hormones, or steroids.

[0011] In certain embodiments, the one or more analytes are ceramide and lyso-sphingomyelin, and the source sample is derived from 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 comprising 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 comprises 0.5% trifluoroacetic acid.

[0012] In some embodiments, 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). In further embodiments, the solvent system is made 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. 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. It should be understood, however, that the detailed description, illustrating embodiments and aspects of the present invention, is given by way of example, not limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram showing the configuration of a conventional online 2D LC-MS / MS. [Figure 2] FIG. 1 compares 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] FIG. 1 is a schematic diagram illustrating the continuous flow 2D LC-MS / MS methodology of the present invention. [Figure 4] 1 shows the analysis of ceramides and lyso-sphingomyelins (lyso-SPMs) using the continuous flow 2D LC-MS / MS methodology of the present invention, in which ceramides and lyso-SPMs were separated from contaminating phospholipids. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present disclosure provides a novel method for simultaneously analyzing multiple analytes in a source sample by 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 phase for both dimensions of the 2D LC-MS / MS analysis. During the 2D LC portion of the analytical run, a diversion valve between the two columns is timed to different positions so that the solvent flow from the first column flows either to waste or to the second column for further separation. Thus, this method allows for the simultaneous analysis of two or more liquid chromatographs. Although a liquid chromatography (LC) column can be used, the need for solvent exchange is avoided, and therefore only one LC pump system (i.e., LC system) is required. 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, e.g., one polar and one nonpolar, as a separation mechanism. As a result, two LC systems are required.

[0016] One major advantage of this method is that it omits the need for a second pumping system, simplifying the subsequent 2D LC-MS / MS configuration (e.g., by using a diverter valve with fewer ports). Eliminating the second pumping system significantly reduces equipment costs and method deployment time. This innovation allows 2D LC-MS / MS analysis to be performed in any laboratory where routine 1D LC-MS / MS analysis is currently performed. This innovation provides flexibility to analytical laboratories, allowing them to choose either 1D or 2D LC-MS / MS analysis using the same equipment. System simplification speeds personnel training, reduces the possibility of assay failure, and shortens sample turnaround time. This method is also amenable to multiplexing, significantly reducing sample preparation and analysis time, as well as sampling bias, and improving reproducibility. Furthermore, the novel 2D-LC approach described herein can be easily adapted to work with detectors other than mass spectrometers, such as charged aerosol detectors (CADs), light scattering detectors, and UV detectors. This flexibility greatly increases the applicability of this analytical method.

[0017] With these improvements, this analytical method will enable the widespread use of 2D LC-MS / MS technology in biochemical analysis for biopharmaceutical research, medical diagnostics, and environmental investigations.

[0018] Sample preparation The present method can be used to analyze (e.g., detect and / or quantify) one or more analytes of interest in any sample matrix, such as a biological or environmental sample. Biological samples can 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, stool, sweat, saliva, bile, tears, cerebrospinal fluid, swabs from body sites, skin, and hair. Environmental samples can be air samples, soil samples, water samples, food samples, and samples of any substance. Analytes of interest can be, for example, small molecules such as drug substances, as well as biomolecules such as polypeptides, peptides, nucleic acids, lipids or fatty acids, carbohydrates, hormones, vitamins, steroids, and metabolites.

[0019] Before applying the sample to the 2D LC system, the analytes of interest can be concentrated and separated by filtration, precipitation, centrifugation, extraction, dilution, or a combination thereof to remove most of the contaminants and interfering substances. For example, solid phase extraction (SPE) can be used to concentrate the components of interest from the source sample. In SPE, a sample preparation cartridge is used to concentrate the analytes of interest. The SPE extract containing the analytes can 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 the present method, the drying and reconstitution steps are not necessary.

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

[0021] Analytes of interest can also be extracted from source samples by solid-supported liquid-liquid extraction (SLE). In SLE, an aqueous solution of the sample is loaded onto a support composed of diatomaceous earth. After sample adsorption onto the support, it is washed several times with an organic extraction solvent, such as methyl tert-butyl ether. The analytes of interest are separated into the organic phase, which is then concentrated by drying and reconstituted in a solvent compatible with 2D LC systems, such as 50:50 methanol:water.

[0022] Proteins may be enriched from source samples by protein precipitation extraction (PPE). Protein precipitation methods may include desalting, isoelectric precipitation, and organic solvent extraction. For example, a source sample is prepared for 2D LC loading by desalting. This protein precipitation method relies on proteins being "salted out" of solution in response to increasing concentrations of neutral salts, such as ammonium sulfate. In another example, a source sample is prepared by isoelectric precipitation. This method may be used to precipitate contaminating proteins rather than the target protein. The isoelectric point (pI) is the pH at which a protein's net primary charge is zero. For most proteins, the pI is in the pH range of 4-6. Inorganic acids, such as hydrochloric acid and sulfuric acid, may also be used as precipitants. A potential drawback of isoelectric precipitation is irreversible denaturation caused by inorganic acids.

[0023] The solvent used to extract and concentrate the analytes of interest from the source sample may be compatible with the 2D LC system. That is, the extraction solvent containing the extracted analytes may be loaded directly onto 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) contains methanol, acetonitrile, and / or water, and the ratio of these three substances can be varied depending on the analyte of interest. For example, to extract lipids from blood samples or tissues, the solvent contains a mixture of methanol, acetonitrile, and water at a total volume percentage of 100%, 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, such as 10 mM ammonium acetate and 1% formic acid. For example, the extraction solvent may contain 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 characteristics of the target analyte and interfering matrix.

[0024] Two-dimensional liquid chromatography For example, a source sample can be treated by concentrating the analytes of interest, and then the treated sample can be input into a liquid chromatography pumping system for application to a first liquid chromatography column.

[0025] Liquid chromatography (LC) is the process of selectively retaining one or more components of a fluid solution (mobile phase) as it passes through a column of finely divided material (stationary phase) by capillary action. Retention of selective components in a fluid solution by the stationary phase is due to the component's greater affinity for the stationary phase than for the mobile phase. As used herein, liquid chromatography includes high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UHPLC), high turbulence liquid chromatography (HTLC), normal phase chromatography (NPC), reversed phase chromatography (RPC), supercritical fluid chromatography (SFC), affinity chromatography, ion exchange chromatography (IEX), capillary liquid chromatography, electrochromatography, membrane chromatography, monolith chromatography, nano- and capillary liquid chromatography. These include, but are not limited to, column chromatography, column chromatography, and size exclusion chromatography (SEC). The analytes of interest may be retained on the stationary phase and subsequently eluted, or may flow through the stationary phase unretained. The analytes in the eluate or effluent can be monitored by various means (e.g., UV, fluorescence, light scattering, or conductivity) based on retention time, peak intensity, and peak area. Further detailed analysis of the analytes 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, the injector is used to introduce the source sample into the mobile phase that transfers it to the LC column. (ii) Reservoir: The solvent reservoir holds the solvent system (mobile phase) used in liquid chromatography separations. (iii) Pump: A high-pressure pump is typically used to generate and maintain a given flow rate of the mobile phase. Traditionally, a dedicated pumping system was required for each LC column. (iv) Column: An LC column contains an inlet port for receiving a sample and an outlet port for discharging the effluent, and is typically packed with a solid adsorbent medium such as silica, polymers, and other resins. LC columns can 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, monolith columns, nano- or capillary LC columns, and chiral chromatography columns. (v) Valves: A selector or switching valve is a high-pressure valve between the column and the next destination of the eluate or effluent passing through the column. The next destination can be, for example, a waste collector or an analyte detector. Diverter (or diverter) valves can be controlled manually or by computer and can optionally 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 interconnect the sample injector, solvent reservoir, pump, column, and detector, and to form the conduit for the mobile phase.

[0027] A 2D LC system has two LC columns with two orthogonal separation mechanisms. The first LC column is the first dimension ( 1 D), and the second LC is called the second dimension ( 2 D). A sample containing one or more target analytes is injected into a first LC column along with a compatible solvent. The solvent passes through the column at high pressure, separating the target analytes 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 The D eluate can be retained on the trap column and then 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 analytes can be further analyzed.

[0028] There are two general types of 2D LC: comprehensive 2D LC (LC×LC), 1 D The total flow of effluent 2 Heart-cutting 2D LC (LC-LC) involves targeting a specific effluent peak or a specific part of the chromatogram. 2 D column. 2 Multiple peaks or multiple portions of the chromatogram can also be selected for transfer to the D column. The LC system's splitter valve allows multiple cuts to be isolated and stored, which can then be transferred to the 2 Analyze on column D.

[0029] Figure 1 shows a typical heart-cutting 2D LC system, with the solid line indicating the mobile phase flow direction. In step 1, a first LC system equipped with a normal-phase (NP) column separates the lipid analytes of interest from interfering phospholipids. A 10-port diverter valve directs the analyte-containing effluent to a trap column, where the analytes are retained, while the phospholipids exit the trap column and are sent to a waste collector. In step 2, a second LC pump system inputs a fluid compatible with a second LC column (reversed-phase or RP). 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 second LC pump inputs the second mobile phase solvent, which passes through the trap column and, depending on the position of the diverter valve, delivers the analytes to the RP column. The analytes are further separated in the RP column and ultimately analyzed by a mass spectrometer.

[0030] The novel 2D LC system of the present disclosure requires only one LC pump system. Flow from the first column to the second column can be continuous without the need for 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., the relative ratio of components) can be adjusted over time as the analytes pass through the separation system. However, because the solvent system is compatible with both columns, no solvent exchange is required as the analytes move from one column to the next. A diverter valve between the two columns can direct the effluent from the first column to waste or to the second column, depending on the expected time of evacuation of the analyte from the first column. Because there is no need for solvent exchange, the diverter valve between the two columns can be simpler, requiring fewer ports. The novel 2D LC system of the present disclosure encompasses both comprehensive 2D LC and heart-cutting 2D LC systems.

[0031] Figure 3 shows a heart-cutting embodiment of the novel 2D LC system of the present disclosure. In step 1, the LC system inputs the sample into the NP column using a solvent system with a first mobile phase ratio, which carries the specific analytes of interest through a three-port diverter valve position to the RP column, where they are retained. In step 2 (i.e., at a later time point), the diverter valve is switched to a second position, directing the NP column effluent, carrying the interfering matrix, to waste. 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 a solvent system with a different mobile phase ratio input 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 LC run contains the same compatible components (although the ratios change 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 (labeled "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 interfering components. In some embodiments, as exemplified above, 1 D column is a normal phase column, 2 The D column is a reversed-phase column. In some embodiments (e.g., for carbohydrate analytes), 1 D column is a normal phase column, 2 The D column is a weak anion exchange column. In some embodiments (e.g., for protein / peptide analytes), 1 D column is a normal phase column, 2 The D column is a reverse-phase column. In some embodiments (e.g., for oligonucleotide analytes such as circulating tumor cell (CTC) DNA), 1 D column is a normal phase column, 2 The D column 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 carbon dioxide. LC solvents include, but are not limited to, hydrogenated solvents (e.g., aliphatic and aromatic solvents), oxygenated solvents (e.g., alcohols, ketones, aldehydes, glycol ethers, esters, and glycol ether esters), and halogenated solvents (e.g., chlorinated and brominated hydrocarbons). LC solvents may be buffered and may include ammonium acetate, ammonium formate, ammonium bicarbonate, acetic acid, trifluoroacetic acid, formic acid, trimethylamine, and triethylamine. In some embodiments, the solvent system used in the present 2D LC system is compatible with the extraction solvent and may include methanol, acetonitrile, and water. In certain embodiments, the solvent system includes about 0-100% methanol, about 0-100% acetonitrile, and about 0-90% water, with the total volume percentage being 100%. In certain embodiments, the solvent system of the 2D LC system is a mixture of various ratios of mobile phase A (mobile phase solvent A) and mobile phase B (mobile phase solvent B), where mobile phase A contains water and 0.5% trifluoroacetic acid, and mobile phase B contains 85% methanol, 15% acetonitrile, and 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 onto 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; less polar lipids elute first and are directed to a reversed-phase column via a diverter valve, while more polar lipids are retained on the NP column. The diversion valve is then switched to the waste position to remove the NP column effluent carrying matrix contaminants. The diversion valve is then returned to its original position, allowing the more polar lipids already retained on the NP column to be transferred to the second dimension for further separation. The timing of the valve switch is adjusted to regulate the solvent flow to either waste or the second column. In the RP column, the more polar lipid analytes are eluted first, while the less polar lipid analytes are retained in the column until a more hydrophobic elution solvent is used. The separated lipid analytes can then be further analyzed by methods such as mass spectrometry.

[0035] Also, by way of example, a sample containing one or more protein / peptide analytes, such as insulin, is extracted with an extraction solvent comprising methanol, acetonitrile, and water. The extraction solvent may further comprise 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 comprising acetonitrile, methanol, and water to separate the protein / peptide analytes from major matrix contaminants (carbohydrates, lipids, nucleotides, etc.). The solvent system may further comprise 0.5% acetic acid and 0.01% trifluoroacetic acid. In some embodiments, the first dimension is a normal-phase column, with more hydrophobic phospholipids eluting first and more hydrophilic polypeptides retained in the normal-phase column. In other embodiments, the first dimension is an anion-exchange column, with positively charged polypeptides eluting first and transferred to the second dimension via a diverter valve, while negatively charged polypeptides are retained on the anion-exchange column. The diverter valve is then switched to waste to remove matrix contaminants. The diversion valve is returned to its original position, transferring 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 waste or the second column, and the design of the solvent gradient can be easily adjusted to separate different protein analytes and eliminate matrix interference. In a further embodiment, the second dimension is a reversed-phase column, and polar proteins are separated. are eluted first, while non-polar proteins are retained within 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. The diversion valve is then switched to waste to remove matrix contaminants. The diversion valve is then switched back to its original position to transfer the nucleic acid analytes already retained in the first dimension to the second dimension for further separation. The timing of valve switching to adjust solvent flow to either the waste or the second column, and the design of the solvent gradient, can be easily adjusted to separate different nucleic acid analytes and eliminate matrix interferences. In a further embodiment, the second dimension is a C18 column, such as a reverse-phase column, using an ion-pairing mechanism. The separated nucleic acid analytes can then be further analyzed, for example, by mass spectrometry.

[0037] Also, by way of example, a sample containing one or more carbohydrate analytes, such as N-linked fetuin 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 onto 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. The diversion valve is then switched to waste to remove the matrix contaminants. The diversion valve is then returned to its original position, and the carbohydrates already retained in the first dimension are transferred 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 not only separate different carbohydrate analytes but also eliminate matrix interferences. In a further embodiment, the second dimension is a weak anion exchange column. The resolved carbohydrate analytes can then be further analyzed, for example, by mass spectrometry.

[0038] Also, by way of 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 then injected onto 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 (e.g., 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. The diversion valve is then switched to waste to remove matrix contaminants. The diversion valve is then returned to its original position, allowing the steroid analytes already retained in the first dimension to be transferred to the second dimension for further separation. The timing of the valve switch 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 eliminate matrix interferences. In a further embodiment, the second dimension is a C18 column, for example a reverse 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 a suitable technique. Mass spectrometry (MS) is often used due to its ultra-high sensitivity. A mass spectrometer ionizes target analytes, separates the ions based on their mass-to-charge ratio in a vacuum, and finally measures the intensity of each ion. Mass spectra are very useful for qualitative and quantitative analysis because they can show the concentration levels of ions with specific masses.

[0040] A mass spectrometer consists of three main components: an ion source for ionizing analytes, a mass analyzer that separates ions based on their mass-to-charge ratio (m / z), and a detector that detects the separated ions. 2The effluent is nebulized, desolvated, and ionized to produce charged particles, or ions. The ions are passed through a series of mass analyzers under vacuum. Precursor ions of a specific (m / z) ratio are selected, and particles of all other (m / z) ratios are filtered out and passed through the mass analyzer. The separated ions are then detected, for example, by an electron multiplier. Sample ionization may be achieved, for example, by electrospray ionization (ESI), atmospheric pressure chemical ionization (ACPI), photoionization, electron impact ionization, chemical ionization, fast atom bombardment (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. Ions may be detected, for example, by multiple reaction monitoring (MRM), selected ion monitoring mode (SIM), selected reaction monitoring (SRM), or an electron multiplier.

[0041] The MS data is sent to a computer, which plots voltage versus time. The concentrations of one or more target analytes in the source sample can be determined by comparing the area under the peak 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 the present disclosure, the mass spectrometry utilizes "tandem mass spectrometry" or "MS / MS," in which selected precursor ions are further fragmented into product ions, for example, by collision with an inert gas such as argon, helium, or nitrogen. A second mass analyzer 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 additional product ion families, and so on. The ion fragmentation pattern is highly specific to the compound's structure, thereby enabling precise structural determination.

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

[0044] The disclosed 2D LC-MS system allows for the purification of target analytes. 2 The system also includes an interface that relays the D column to the mass spectrometer. This interface is used due to the inherent incompatibility between liquid chromatography and mass spectrometry. The mobile phase solvent system in an LC system is a pressurized fluid, whereas MS instruments typically operate under vacuum. This interface transfers the target analytes from the LC unit to the MS unit, removing most of the mobile phase solvent system used in the liquid chromatography separation process and preserving the chemical identity of the target analytes.

[0045] In some embodiments, the interface is an electrospray interface. Alternatively, the interface is an atmospheric pressure ionization interface, an atmospheric pressure chemical ionization interface, a thermospray interface, a moving belt interface, or the like. The interface may be a direct liquid introduction interface, a particle beam interface, or a fast atom bombardment (FAB) based interface.

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

[0047] application Novel 2D LC-MS systems, including the novel 2D LC-MS / MS systems of the present disclosure, can be used to analyze (including detect and quantify) a variety of molecules, including small molecules (e.g., drug substances) 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 and development for screening / diagnostic and therapeutic purposes. For example, the 2D LC-MS / MS system may be used to measure the 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 described in the Examples below, a 2D LC-MS / MS system can be used to analyze two lipid biomarkers: ceramide (CER) and lyso-sphingomyelin (lyso-SPM) in blood samples from ASMD patients. ASMD is a sphingolipid metabolic disorder that results in the accumulation of sphingomyelin in tissues throughout the body, particularly the spleen, liver, lungs, bone marrow, and occasionally the brain. Elevated levels of lyso-SPM (sphingosinephosphocholine) are also common. The defective enzyme in these patients, acid sphingomyelinase, catalyzes the hydrolysis of sphingomyelin in lysosomes to produce phosphocholine and ceramide. Lipids such as ceramide and lyso-SPM are highly elevated in ASMD patients and can therefore be used as biomarkers for the screening and diagnosis of ASMD and to monitor enzyme replacement therapy (using recombinant human ASMs such as lysoprotease alpha).

[0050] In order that this invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and are not to be construed as in any way limiting the scope of the invention. [Example]

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

[0052] Chemicals and Reagents Standard reference ceramide (from porcine brain) was purchased from Avanti Polar Lipids (AL, USA). CER internal standard (N-nonadecanoyl-D-erythro-sphingosine) and lyso-sphingomyelin standard (sphingosylphosphorylcholine) were purchased from Matreya (PA, USA). Lyso-SPM internal standard (d9-lysosphingomyelin) was synthesized in-house. Methanol and acetonitrile (both HPLC grade) were purchased from Honeywell (NC, USA). Ammonium acetate was used. Monomethanol was purchased from Sigma Aldrich (MO, USA). Deionized water was obtained using an in-house 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 working solution (80% methanol, 20% acetonitrile, 10 mM ammonium acetate, and 1% formic acid) containing CER internal standard (IS) (0.22 μg / mL) in a 96-well plate. The mixture was vortexed at 11,000 rpm for 10 minutes using a DVX-2500 multitube 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 an antiparallel 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 concentrations in each sample were quantified as μg / mL, and lyso-SPM was quantified as ng / mL in plasma based on a calibration curve using a 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 setup 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 an 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 setup included the following connections: a) PEEK tubing (approximately 60 cm long) connecting the outlet of the first-dimension analytical column to port 1 of the diversion valve b) A second PEEK tube (approximately 70 cm long) connecting port 2 of the diverter valve to the inlet of the second-dimension analytical column. c) a third PEEK tube (approximately 80 cm long) connecting the outlet of the second-dimensional analytical column to the mass spectrometer; d) A fourth PEEK tube (at least 60 cm long) connecting port 3 of the diverter valve to waste.

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

[0057] [Table 1]

[0058] LC Diversion Valve Conditions In conjunction with the solvent system adjustments shown in the table above, the 2D LC system was switched to different port positions at predetermined times as shown in Table 2 below. 1Different fractions of the D effluent could be directed either to waste or to the next (RP) column, see also Figure 4.

[0059] [Table 2]

[0060] mass spectrometry For the analysis of ceramides and lyso-SPMs, we used a multiple reaction monitoring (MRM) technique. This technique uses a triple quadrupole MS to first target ions corresponding to the compounds of interest, ceramides and lyso-SPMs, and then fragments the target ions to generate various daughter ions. One (or more) of these fragment daughter ions are selected for quantification. Only compounds that meet both of these criteria, i.e., a specific parent ion and a specific daughter ion corresponding to the compound's mass, are separated in 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 MRM channel parameters for the ceramide internal standard (IS), lyso-SPM analytes, and lyso-SPM IS.

[0064] [Table 4]

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

[0066] More specifically, in this LC process, the diversion valve was first set to position A to allow for column flushing. Then, the valve was set to position B, and ceramide was separated from phospholipids in the normal phase (NP) column and flowed into and retained in the RP column. The diversion valve was then switched to waste (position A), 1 The phospholipids and other matrix components following the D effluent were removed. Finally, the diversion valve was switched back to position B, allowing the lyso-SPMs to enter the RP column, and both ceramides and lyso-SPMs were separated from other potentially interfering molecules prior to mass analysis.

[0067] The data demonstrate 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. The phospholipids were diverted to waste by setting the diversion valve to position A, and the ceramide and lyso-SPM analytes were directed to the RP column by setting the valve to position B. On the RP column, lyso-SPM appeared as a single peak, while the ceramide analytes fractionated into distinct retention windows, including the following isoforms depending on the length of the fatty acid chain and the number of double bonds: C16, C18, C20 and C22:1, C22 and C24:1, C24, and C26:1. The data demonstrate that CER isoforms eluted from the RP column as sharp peaks with little 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 turnaround time for quantifying CER and lyso-SPM in human plasma from typically 8 days to just 2 days. Furthermore, the original labor-intensive offline extraction process, including SPE and protein precipitation (PPT) procedures for sample cleanup, was reduced from 25 hours to 1 hour by utilizing the extraction solvent and continuous-flow 2D LC of the present invention. See, e.g., 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 to qualify a 2D LC-MS / MS system for detecting ceramide. In this study, samples, calibration curve standards, and controls were added to a 96-well plate. Next, an assay working solution containing a known amount of internal standard (CER-IS) was added to the wells. The plate was then vortexed and analyzed as described in Example 1. The mixture was sonicated and centrifuged. After centrifugation, the supernatant was transferred to a new 96-well plate. The 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 the individual CER isoforms were integrated using Analyst™ software. The peak areas of the individual CER isoforms for any given sample were then summed in Excel. The total CER peak area was 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] The novel multiplex assay was qualified for CER quantification. Qualification parameters included system suitability, intra- and inter-run precision and accuracy, linearity of the calibration (standard) line, analytical sensitivity, carryover, delipidated plasma matrix effect, reinjection reproducibility, batch size assessment, extraction recovery, completeness of sample dilution, determination of CER standards in healthy donors, and comparison of methods for determining plasma CER levels.

[0073] Carryover was assessed by injecting solvent (assay working solution only) after the highest calibration standard of each run. Defatted plasma matrix effects were evaluated using three independent lots of delipidated plasma spiked with three different concentrations of analyte: low quality control (LQC), medium quality control (MQC), and high quality control (HQC). Reinjection reproducibility was assessed by continuously reinjecting samples at LLOQ (lower limit of quantification), LQC, MQC, HQC, and ULOQ (upper limit of quantification) concentrations. Extraction recovery was assessed by comparing two pooled patient samples before and after the addition of CER. The internal standard (IS) was introduced at the same stage for both pre- and post-addition conditions.

[0074] This data demonstrates that our novel 2D LC-MS / MS method detected 11 CER isoforms by multiple reaction monitoring (MRM) using independent MS / MS channels for each of these isoforms. System suitability testing demonstrated %CVs between 0% and 5% for all pass runs performed throughout the validation process. The mean %Bias and mean %CV (coefficient of variation) for all concentrations met established criteria 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 mean %bias at the LLOQ were 8% and -8%, respectively.

[0075] The minimum and maximum carryover of the analyte peak areas 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 the reinjection repeatability was 2% to 4% at a given concentration. The recoveries of the extracts ranged from 67% to 88%, with %CVs varying from 2% to 11%. The results demonstrated that the method is precise and accurate and is suitable for use in measuring ceramide concentrations in human plasma.

Claims

1. 1. A method for monitoring one or more lipid biomarkers in a blood sample of a subject in need thereof, comprising the steps of: (a) extracting one or more lipid biomarkers from a blood sample with an extraction solvent to obtain an extracted sample; (b) applying the extracted sample and solvent system to a first liquid chromatography (LC) column equipped with a liquid chromatography (LC) pumping system, the first LC column being directly connected to a second LC column via tubing equipped with a diverter valve; (c) setting the diversion valve to a first position at a first predetermined time so that solvent effluent from the first LC column is directed to waste; (d) setting the diversion 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) analyzing the chromatographically separated sample obtained from the second LC column; Including, The method utilizes only one LC pump system for both LC columns. The method.

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

3. The method of claim 2 , wherein the MS is tandem MS.

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

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

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

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

8. 8. The method of any one of claims 1 to 7, wherein the relative ratios of the components of the solvent system are varied during a single sample run.

9. 9. The method of any one of claims 1 to 8, wherein the subject has or is at risk of developing a lysosomal storage disease.

10. 10. The method of claim 9, wherein the lysosomal disease is Fabry disease, Gaucher disease, Krabbe disease, and acid sphingomyelinase deficiency (ASMD).

11. 11. The method of claim 10, wherein the subject is being treated with or in need of enzyme replacement therapy.

12. 12. The method of claim 11, wherein the lysosomal disease is ASMD.

13. The method of claim 12, wherein the one or more lipid biomarkers include ceramide and / or lyso-sphingomyelin analytes.

14. 14. The method of claim 13, wherein the one or more lipid biomarkers are extracted from the 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.

15. The method of any one of claims 9 to 14, wherein the first LC column is a silica column and the second LC column is a C18 column.

16. 16. The method of claim 15, wherein the solvent system comprises 0.5% trifluoroacetic acid.

17. 17. The method of claim 15 or 16, 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).

18. 18. The method of claim 17, 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.

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

1.

20. 10. The method of claim 1, comprising repeating steps (c) and (d).

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