Two-dimensional LC-MS / MS system

Abstract

The invention relates to a two-dimensional LC-MS / MS system. A novel analytical method for detecting one or more analytes in a source sample using a single LC system by continuous flow 2DLC-MS / MS is provided.

Description

[0001] This invention application is a divisional application based on an invention patent application with an application date of May 29, 2020, application number 202080040211.1 (international application number PCT / US2020 / 035231) and name “Two-dimensional LC-MS / MS system”. [Technical field]

[0002] This application claims priority to U.S. Provisional Application No. 62 / 855,636, filed on May 31, 2019, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0003] Liquid chromatography tandem mass spectrometry (LC-MS / MS) emerges as a powerful technique for detecting and measuring a wide variety of analytes. It quickly becomes a method selection for accurately and precisely quantifying analytes from biological matrices. The main obstacle to LC-MS / MS analysis of samples from biological matrices is the presence of interference with the identification and quantification of the analytes of interest present in these matrices. The main matrix components, such as phospholipids, proteins or nucleotides (see, for example, Carmical and Brown, Biomed. Chromatogr. A (2016) 30: 710-20; and Massood et al., Lipids (2012) 47: 209-26). The basic steps of developing bioanalytical methods require cleaning samples to eliminate major matrix impurities before sample analysis. Methods for sample cleaning are broadly classified as either "offline" (which refers to a manual sample preparation procedure) or "online" (which refers to a sample preparation procedure performed by a liquid chromatography (LC) system) (Afonso-Olivares et al., J Chromatogr. (2017) 1487:54-63; Mokh et al., Sci Total Environ. (2017) 609:830-41; and Dong et al., Bioanalysis (2015) 7:2227-33).

[0004] One of the most common offline sample cleanup methods is solid phase extraction (SPE), which focuses on eliminating matrix components and enriching the analytes of interest using sample preparation cartridges (Vanol et al., Biomed. Chromatogr. (2017) 32: 1-10; see also Afonso-Olivares, Mokh and Dong, supra). The SPE extract containing the analytes of interest is dried and reconstituted in another solvent system, which needs to be compatible with one-dimensional (1D) LC-MS / MS analysis. The method of drying and reconstituted samples in different solvent systems is called "solvent exchange" or "buffer exchange". This process is necessary because the solvent system used for SPE is generally not compatible with the solvent system used for 1D LC-MS / MS analysis. The solvent exchange process results in sample loss and reduced reproducibility. In addition, SPE cartridges are costly and the process is time-consuming. In order to eliminate the SPE step for sample cleanup, online two-dimensional (2D) LC-MS / MS has been developed. 2D LC-MS / MS uses a dual column system. The first column, usually made of a hydrophilic resin (normal phase chromatography), is used to eliminate major matrix impurities before the sample of interest is introduced into the second column. The second column can be made of a hydrophobic resin (reverse phase chromatography) and further resolves the analyte of interest from other interfering molecules before introduction into the mass spectrometer (Iguiniz and Heinisch, J Pharmaceut Biomed Anal. (2017) 145: 482-503; Ling et al., Biomed. Chromatogr. (2014) 28: 1284-93; Pirok et al., Anal Chem. (2019) 91: 240-63; and Iguiniz et al., Talanta. (2019) 195: 272-80). Therefore, for traditional online 2D LC-MS / MS analytical methods, two different solvent systems are used. This requires a dedicated pump system for each column and a multi-port diverter valve that allows analyte transfer and solvent exchange. These requirements make traditional 2D LC-MS / MS methods extremely complex and expensive, outweighing the potential benefits of 2D LC-MS / MS. Most laboratories are not equipped to perform this type of analysis.

[0005] Therefore, there remains a need for improved analytical methods that are accurate yet simple. [Summary of the invention]

[0006] The present disclosure provides a novel 2D LC analysis method for analyzing one or more analytes in a source sample. The method comprises the following steps: (a) extracting the one or more analytes from the source sample using an extraction solvent to obtain an extracted sample; (b) applying the extracted sample and the solvent system to a first liquid chromatography (LC) column using an LC system, wherein the first LC column is directly connected to a second LC column via a cannula having a diverter valve; (c) setting the diverter 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 diverter 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 needed; and (f) analyzing a sample obtained from the chromatographic separation of the second LC column. An LC system refers to an LC instrument having a pump system for inputting a solvent system into an LC column. By "directly" is meant that there is no second LC system dedicated to the second column, and thus the methods of the invention utilize only one LC system for the second dimension. 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 of 2D LC comprises one or more of methanol, acetonitrile and water. In other embodiments, the solvent system further comprises ammonium acetate and / or formic acid.

[0009] By "solvent system" is meant the solvent mixture or combination used during an LC run for analyzing the target analyte. During a run, the solvent composition may change (e.g., by changing the relative ratios of the components of the solvent mixture), but the change does not necessitate a solvent exchange of the sample as it travels from the first column to the second column. In some embodiments, the relative ratios of the components of the solvent system vary during a 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, the one or more analytes are proteins, lipids, carbohydrates, nucleotides, metabolites, vitamins, hormones or steroids.

[0011] The one or more analytes are ceramide and lyso-sphingomyelin, and the source sample is blood from a patient with acid sphingomyelinase deficiency. In further embodiments, the ceramide and the 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. In particular 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 having 18 carbons). In further embodiments, the solvent system comprises 0.5% trifluoroacetic acid.

[0012] In some embodiments, the solvent system applied to the first LC column and the second LC column comprises 0-85% methanol (v / v), 0-15% acetonitrile (v / v), and 0-100% water (v / v). In other embodiments, the solvent system is made by mixing a first solvent comprising water and 0.5% trifluoroacetic acid and 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, objectives and advantages of the present invention are apparent in the following embodiments. However, it should be understood that the embodiments, although indicating embodiments and aspects of the present invention, are provided by way of illustration and not limitation. For those skilled in the art, various changes and modifications within the scope of the present invention will become apparent from the embodiments.

[0014] Specifically, this application includes but is not limited to the following:

[0015] 1. A method for analyzing one or more analytes in a source sample, comprising the following steps:

[0016] (a) extracting the one or more analytes from the source sample using an extraction solvent to obtain an extracted sample;

[0017] (b) applying the extracted sample and the solvent system to a first liquid chromatography (LC) column using an LC system, wherein the first LC column is directly connected to a second LC column via a cannula having a diverter valve;

[0018] (c) setting the diverter valve to a first position at a first predetermined time so that solvent effluent from the first LC column is directed to waste;

[0019] (d) setting the diverter 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;

[0020] (e) repeating steps (c) and (d) as necessary; and

[0021] (f) analyzing a sample obtained from the chromatographic separation of the second LC column.

[0022] 2. The method of claim 1 , wherein step (f) comprises analyzing the chromatographically separated sample obtained from the second LC column using mass spectrometry (MS).

[0023] 3. The method of item 2, wherein the MS is a tandem MS.

[0024] 4. The method of any of the preceding items, wherein the LC is high performance liquid chromatography or ultra high performance liquid chromatography.

[0025] 5. The method of any of the preceding items, wherein the first LC column is a normal phase column and the second LC column is a reverse phase column, or vice versa.

[0026] 6. The method of any of the preceding items, wherein the solvent system comprises one or more of methanol, acetonitrile and water.

[0027] 7. A method as described in claim 6, wherein the solvent system further comprises ammonium acetate and / or formic acid.

[0028] 8. The method of any of the preceding clauses, wherein the relative ratios of the components of the solvent components of the solvent system are varied during a single sample run.

[0029] 9. The method of any of the preceding items, wherein the source sample is a biological sample.

[0030] 10. The method of 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.

[0031] 11. The method of claim 9 or 10, wherein the one or more analytes are proteins, lipids, carbohydrates, nucleotides, metabolites, vitamins, hormones or steroids.

[0032] 12. The method of claim 11, wherein the one or more analytes are ceramide and lyso-sphingomyelin, and the source sample is blood from a patient suffering from acid sphingomyelinase deficiency.

[0033] 13. A method as described in claim 12, wherein the ceramide and the lyso-sphingomyelin are extracted from a blood sample using an extraction solvent, and the extraction solvent comprises 80% methanol (v / v), 15-20% acetonitrile (v / v), 0-5% water (v / v), 10 mM ammonium acetate and 1% formic acid.

[0034] 14. The method of claim 12 or 13, wherein the first LC column is a silica column and the second LC column is a C18 column.

[0035] 15. A method as described in claim 14, wherein the solvent system comprises 0.5% trifluoroacetic acid.

[0036] 16. The method of claim 14 or 15, wherein the solvent system applied to the first LC column and the second LC column comprises 0-85% methanol (v / v), 0-15% acetonitrile (v / v) and 0%-100% water (v / v).

[0037] 17. A method as described in claim 16, wherein the solvent system can be obtained by mixing a first solvent comprising water and 0.5% trifluoroacetic acid and a second solvent comprising 85% methanol (v / v), 15% acetonitrile (v / v) and 0.5% trifluoroacetic acid.

[0038] 18. A method as described in claim 17, wherein the ratio of the first solvent to the second solvent is 70:30, 85:15 or 99:1.

Brief Description of the Drawings

[0039] Figure 1 Schematic diagram showing the setup of a conventional on-line 2D LC-MS / MS.

[0040] Figure 2 Comparison of the settings for off-line 1D LC-MS / MS, traditional on-line 2D LC-MS / MS, and current on-line continuous-flow 2D LC-MS / MS (single LC system).

[0041] Figure 3 Schematic representation illustrating the current continuous-flow 2D LC-MS / MS method.

[0042] Figure 4 The analysis of ceramide and lyso-sphingomyelin (lyso-SPM) using the current continuous flow 2D LC-MS / MS method is demonstrated. Ceramide and lyso-SPM are separated from contaminating phospholipids in this method. [Implementation Method]

[0044] 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 (2DLC-MS / MS). In current 2DLC-MS / MS methods, a single solvent system is used as an extraction solvent and a mobile phase for two dimensions of 2D LC-MS / MS analysis. During the 2D LC portion of the analytical run, the diverter valve between the two columns is switched to different positions in a timed manner to direct the solvent flow from the first column to the waste or the second column for further separation. Therefore, when two or more liquid chromatography (LC) columns are allowed to be used, this method only requires one LC pump system (i.e., LC system) because the method bypasses the need for solvent exchange. In addition, the solvent flow in the method of the present invention is continuous, without any solvent exchange step or second LC system. In contrast, conventional 2DLC-MS / MS utilizes two separation mechanisms with two different solvent systems (e.g., one polar and the other non-polar). Therefore, two LC systems are required.

[0045] A great advantage of the method of the present invention is the elimination of the second pump system and the subsequent simplification of the 2D LC-MS / MS setup (e.g., using a diverter valve with fewer ports). The elimination of the second pump system greatly reduces equipment costs and method development time. This innovation allows 2D LC-MS / MS analysis to be performed in any laboratory that is currently performing conventional 1D LC-MS / MS analysis. This innovation adds flexibility to analytical laboratories, allowing them to choose to perform 1D or 2D LC-MS / MS analysis using the same test equipment. The simplification of the system makes personnel training faster, reduces the probability of analytical failures, and reduces sample turnaround time. This method is also suitable for multitasking, which can significantly reduce sample preparation and analysis time and sampling deviations, and improve reproducibility. In addition, the novel 2D-LC method described herein can be easily adapted to work on detectors other than mass spectrometers, such as charged aerosol detectors (CAD), light scattering detectors, or UV detectors. This flexibility greatly increases the applicability of current analytical methods.

[0046] Due to these improvements, the current analytical methods will allow 2D LC-MS / MS technology to be widely used in the field of biochemical analysis for use in biomedical research, medical diagnostics, and environmental research.

[0047] Sample preparation

[0048] The methods of the present invention 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 living organelles, such as cell and tissue cultures, tissue sections, 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 can be air samples, soil samples, water samples, food samples, and any material samples. Analytes of interest can be, for example, small molecules, such as drug substances; and biomolecules, such as polypeptides, peptides, nucleic acids, lipids or fatty acids, carbohydrates, hormones, vitamins, steroids, and metabolites.

[0049] In order to remove most of the impurities and interfering materials before applying the sample to the 2D LC system, the analyte of interest can be enriched and separated by filtration, precipitation, centrifugation, extraction, dilution or a combination thereof. By way of example, the analyte of interest is enriched from the source sample by solid phase extraction (SPE). SPE enriches the analyte of interest by using a sample preparation cartridge. The SPE extract containing the analyte can be dried and restored in a solvent system compatible with the 2D LC system. If the SPE extraction solvent is compatible with the 2D LC system, then, as in some embodiments of the method of the present invention, no drying and restoration steps are required.

[0050] 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 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, after which it is extracted, concentrated, and diluted.

[0051] The analytes of interest can also be extracted from the source sample by solid supported liquid-liquid extraction (SLE). In SLE, an aqueous solution of the source sample is loaded onto a support comprising diatomaceous earth. After the sample is absorbed into the support, it is washed several times with an organic extraction solvent such as methyl tert-butyl ether. After the analytes of interest have been partitioned into the organic phase, they are concentrated by drying before being reconstituted into a solvent compatible with the 2D LC system, such as a 50:50 methanol:water solution.

[0052] If the analyte of interest is a protein, it can also be enriched from a source sample by protein precipitation extraction (PPE). Protein precipitation methods may include desalting, isoelectric precipitation, and organic solvent extraction. By way of example, a source sample is prepared for 2D LC loading by desalting. This protein precipitation technique relies on the "salting out" of a protein from a solution in response to an increase in the concentration of a neutral salt such as ammonium sulfate. In another embodiment, the source sample is prepared by isoelectric precipitation; this method can be used to precipitate impurity proteins rather than target proteins. The isoelectric point (pI) is the pH at which the net primary charge of a protein becomes zero. For most proteins, the pI is in the pH range of 4-6. Mineral acids such as hydrochloric acid and sulfuric acid can be used as precipitants. A potential disadvantage of isoelectric precipitation is the irreversible denaturation caused by the inorganic acid.

[0053] The solvent used for extracting from the source sample and enriching the analyte of interest can be compatible with the 2D LC system. That is, the extraction solvent containing the extracted analyte can be directly loaded into the 2D LC system without the need for solvent exchange. In some embodiments, the extraction solvent of biomolecules (e.g., polypeptides, peptides, nucleic acids, lipids, hormones, vitamins, steroids and carbohydrates) comprises methanol, acetonitrile and / or water, wherein the ratio of these three substances can be different depending on the analyte of interest. For example, in order to extract lipids from blood samples or tissues, the solvent comprises a mixture of 100% methanol, acetonitrile and water, such as about 30-100% methanol (v / v), about 0-100% acetonitrile (v / v) and about 0-50% water (v / v). Optionally, the solvent may contain other ingredients, such as 10mM 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 may contain 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 target analyte and the interfering matrix characteristics.

[0054] 2D-LC

[0055] Once the source sample has been processed, for example, by enriching the analyte of interest, the processed sample may be input into a liquid chromatography pump system for application to a first liquid chromatography column.

[0056] Liquid chromatography (LC) is a process that selectively retains one or more components of a fluid solution as it permeates through a column of finely divided material (stationary phase) by capillary action. The retention of selective components in a fluid solution by a stationary phase results from the component having a higher affinity for the stationary phase than for the mobile phase. Liquid chromatography as used herein includes, but is not limited to, high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC), high turbulence 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, monolith chromatography, nano- and capillary liquid chromatography, and size exclusion chromatography (SEC). The analyte of interest may be retained by the stationary phase and subsequently eluted, or may flow through the stationary phase without being retained. 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. A more detailed analysis of the analytes can be performed using techniques such as mass spectrometry as described below.

[0057] Liquid chromatography (LC) systems typically contain some or all of the following components.

[0058] (i) Injector: Also known as a sample manager or autosampler, an injector is used to introduce a source sample into the mobile phase which transfers it to the LC column.

[0059] (ii) Reservoir: The solvent reservoir contains the solvent system (mobile phase) used for liquid chromatography separation.

[0060] (iii) Pumps: High-pressure pumps are usually used to generate and maintain a specified flow rate of the mobile phase. Traditionally, each LC column requires a dedicated pump system.

[0061] (iv) Column: An LC column includes an inlet port for receiving a sample and an outlet port for releasing an effluent, and is typically packed with a solid adsorbent medium such as silica, polymers, and other resins. LC columns may be, for example, normal phase columns (typically hydrophilic), reverse phase columns (typically hydrophobic), cation exchange columns, anion exchange columns, size exclusion chromatography columns, membrane columns, monolithic columns, nano- or capillary LC columns, and chiral chromatography columns.

[0062] (v) Valves: A splitter or 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 collector or an analyte detector. A splitter (or diverter) valve may be controlled manually or by a computer and may optionally include a trapping column. Typically, a diverter valve used in an LC system may have up to 12 ports.

[0063] (vi) Accessories: High-pressure bushings and fittings are used to interconnect sample syringes, solvent reservoirs, pumps, columns and detectors to form the tubing for the mobile phase.

[0064] A 2D LC system has two LC columns with two orthogonal separation mechanisms. The first LC column is referred to as the first dimension (1D), and the second LC is referred to as the second dimension (2D). A sample containing one or more target analytes is injected into a first LC column with a compatible solvent. The solvent flows through the column at high pressure, where the target analyte is separated from impurities in the sample. The effluent from the first LC column is collected and injected into the second LC column, where the target analyte is further resolved. Alternatively, the 1D eluent may be retained in a capture column before being injected into the second dimension. A guard column containing a stationary phase similar to that of a 2D column may be used as a capture column. After elution from the second dimension, the effluent containing the target analyte may be further analyzed.

[0065] There are generally two types of 2D LC. In full 2D LC (LC×LC), the entire stream of the 1D effluent is directed to a 2D column. In heart-cut 2D LC (LC-LC), a specific effluent peak or a specific portion of the chromatogram is directed to a 2D column. Multiple peaks or portions of the chromatogram can also be selected for transfer to a 2D column. Diverter valves in the LC system allow multiple cuts to be cut and stored, which are then analyzed in a 2D column.

[0066] Figure 1 Illustration of a typical heart-cutting 2D LC system, where the solid line indicates the direction of flow of the mobile phase. In step 1, a first LC system with a normal phase (NP) column separates the lipid analyte of interest from interfering phospholipids. A 10-port diverter valve directs the effluent containing the analyte to a trapping column, where the analyte is retained by the trapping column, while the phospholipids leave the trapping column and continue to a waste collector. In step 2, a second LC pump system inputs a fluid that is compatible with the second LC column (reversed phase or RP); by controlling the position of the 10-port valve, this fluid flows through the trapping column, thereby achieving solvent exchange in the trapping column. In step 3, a second LC pump inputs a second mobile phase solvent, which runs through the trapping column and brings the analyte to the RP column via the positioning diverter valve. The analyte is further separated by the RP column and ultimately analyzed by mass spectrometry.

[0067] In the novel 2D LC system of the present disclosure, only one LC pump system is required. In the case where a solvent exchange step is not required, the flow from the first column to the second column can be continuous. This is possible because a mobile phase solvent system is used. Although the composition of the mobile phase solvent system (e.g., the relative ratio of the components) can be adjusted in a timed manner when the analyte travels through the separation system, there is no need to perform solvent exchange when the analyte moves from one column to the next column, because even when adjusting its composition, the solvent system is compatible with the two columns. Depending on the expected time for the analyte to leave the first column, the diverter valve between the two columns can direct the effluent from the first column to waste or to the second column. Because there is no need to perform solvent exchange, the diverter valve between the two columns can be simpler, thereby requiring fewer ports. The novel 2D LC system of the present disclosure covers both full 2D LC and heart-cut 2D LC systems.

[0068] Figure 3 A heart-cutting embodiment of a novel 2D LC system of the present disclosure is illustrated. In step 1, the LC system inputs a sample through a solvent system having a first mobile phase ratio with a NP column, wherein, via a positioned three-port diverter valve, this solvent system carries certain analytes of interest to a RP column, where the analytes of interest are retained in the RP column. In step 2 (i.e., at a later point in time), the diverter valve is switched to a second position such that the NP column effluent carrying interfering matrix is ​​directed to waste. In step 3, additional analytes from the NP column are passed to the RP column, and all target analytes retained by the RP column are eluted through a solvent system having 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 used for the entire LC run contains the same compatible components (although in different ratios as the run proceeds), there is no need to interrupt the solvent flow for solvent exchange. Furthermore, in contrast to conventional 2D LC systems, only one pumping system (shown as "LC System 1") is required.

[0069] The 1D and 2D columns in the current novel system can be selected based on the properties of the target analyte and the matrix interfering components. In some embodiments, the 1D column is a normal phase column and the 2D column is a reverse phase column, as described above. In some embodiments (e.g., for carbohydrate analytes), the 1D column is a normal phase column and the 2D column is a weak anion exchange column. In some embodiments (e.g., for protein / peptide analytes), the 1D column is a normal phase column and the 2D column is a reverse phase column. In some embodiments (e.g., for oligonucleotide analytes, such as circulating tumor cell (CTC) DNA), the 1D column is a normal phase column and the 2D column is a reverse phase column with an ion pairing reagent.

[0070] LC solvents may include, but are not limited to, water, methanol, ethanol, acetonitrile, trifluoroacetic acid, heptafluorobutyric acid, ethers, hexane, ethyl acetate, and organic solvents such as hydrocarbon 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 contain ammonium acetate, ammonium formate, ammonium bicarbonate, acetic acid, trifluoroacetic acid, formic acid, trimethylamine, and triethylamine. In some embodiments, the solvent system used in the current 2DLC system is compatible with the extraction solvent and may contain methanol, acetonitrile, and water. 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 a specific embodiment, 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) at various ratios, wherein mobile phase A contains water and 0.5% trifluoroacetic acid, and mobile phase B contains 85% methanol, 15% acetonitrile, and 0.5% trifluoroacetic acid.

[0071] By way of example, a source sample comprising one or more lipid analytes (e.g., sphingolipids, cholesterol, and triglycerides) is extracted with an extraction solvent comprising 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 then loaded onto an LC system using a mobile phase solvent system comprising acetonitrile, methanol, and water, where the lipid analytes are separated from major matrix impurities (carbohydrates, proteins, nucleotides, etc.). The solvent system may further comprise 10 mM ammonium acetate, 1% formic acid, and 0.5% trifluoroacetic acid. The first LC column is an NP column, where the less polar lipids elute first and are directed to a reverse phase column via a diverter valve, while the more polar lipids are retained in the NP column. The diverter valve is then switched to the waste position for elimination of the NP column effluent carrying the matrix impurities. The diverter valve is then switched back to the original position to transfer the still more polar lipid analytes previously retained in the NP column to the second dimension for further analysis. The timing valve is switched to regulate the solvent flow to the waste or 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 resolved lipid analytes can then be further analyzed, for example, by mass spectrometry.

[0072] Also by way of example, a sample comprising one or more protein / peptide analytes (e.g., 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, wherein the protein / peptide analytes are separated from major matrix impurities (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, wherein more hydrophobic phospholipids are eluted first, while more hydrophilic polypeptides are retained in the normal phase column. In some other embodiments, the first dimension is an anion exchange column, wherein positively charged polypeptides are eluted first and transferred to the second dimension via a diverter valve, while negatively charged polypeptides are retained in an anion exchange column. Then, the diverter valve is switched to waste to eliminate matrix impurities. The diverter valve is switched back to the original position to transfer the polypeptides previously retained in the first dimension into the second dimension for further analysis. The timing valve is switched to adjust the solvent flow to the waste or the second column, and the solvent gradient design can be easily adjusted to separate different protein analytes and eliminate matrix interference. In other embodiments, the second dimension is a reverse phase column, where polar proteins elute first, while non-polar proteins are retained in the column until a more hydrophobic elution solvent is used. The resolved protein analytes can then be further analyzed, for example, by mass spectrometry.

[0073] Also by means of embodiments, a sample comprising one or more nucleic acid analytes (e.g., synthetic oligonucleotides) is extracted with an extraction solvent comprising water and acetonitrile. The extracted sample is then injected into a silica column (e.g., a normal phase column) using a mobile phase solvent system comprising methanol, acetonitrile and water, wherein the nucleic acid analytes are separated from major matrix impurities (carbohydrates, lipids, proteins, etc.). The solvent system may further comprise trimethylamine or triethylammonium bicarbonate. Then, the diverter valve is switched to waste to eliminate matrix impurities. The diverter valve is switched back to the original position to transfer the nucleic acid analytes previously retained in the first dimension to the second dimension for further analysis. The timing valve is switched to regulate the flow of solvent to waste or the second column, and the solvent gradient design can be easily adjusted to separate different nucleic acid analytes and eliminate matrix interference. In other embodiments, the second dimension is a C18 column using an ion pairing mechanism, such as a reverse phase column. The nucleic acid analytes resolved can then be further analyzed, for example, by mass spectrometry.

[0074] Also by way of example, a sample comprising one or more carbohydrate analytes (e.g., N-linked fetuin oligosaccharides) is extracted with an extraction solvent comprising methanol, acetonitrile and water. The extraction solvent may further comprise 10 mM ammonium acetate, 0.5% acetic acid and 0.1% formic acid. The extracted sample is then injected into a silica column (e.g., a normal phase column) using a mobile phase solvent system comprising methanol, acetonitrile and water, wherein the carbohydrate analytes are separated from the main matrix impurities (lipids, proteins, nucleic acids, etc.). The solvent system may further comprise 10 mM ammonium acetate, 1% formic acid and 0.5% trifluoroacetic acid. Then, the diverter valve is switched to waste to eliminate matrix impurities. The diverter valve is switched back to the original position to transfer the carbohydrate analytes previously retained in the first dimension to the second dimension for further analysis. The timing valve is switched to adjust the solvent flow to the waste or the second column, and the solvent gradient design can be easily adjusted to separate different carbohydrate analytes and eliminate matrix interference. In another embodiment, the second dimension is a weak anion exchange column. The resolved carbohydrate analytes may then be further analyzed, for example, by mass spectrometry.

[0075] Also by means of an embodiment, a sample comprising one or more steroid analytes (e.g., dihydrotestosterone) is extracted with an extraction solvent comprising methanol, acetonitrile and water. The extraction solvent may further comprise 10 mM ammonium acetate, 0.5% acetic acid and 1% formic acid. The extracted sample is then injected into a silica column (e.g., a normal phase column) using a mobile phase solvent system comprising methanol, acetonitrile and water, wherein the steroid analytes are separated from the main matrix impurities (carbohydrates, lipids, proteins, nucleic acids, etc.). The solvent system may further comprise 10 mM ammonium acetate, 0.5% acetic acid and 0.5% trifluoroacetic acid. Then, the diverter valve is switched to waste to eliminate matrix impurities. The diverter valve is switched back to the original position to transfer the steroid analytes previously retained in the first dimension to the second dimension for further analysis. The timing valve is switched to adjust the solvent flow to the waste or the second column, and the solvent gradient design can be easily adjusted to separate different steroid analytes and eliminate matrix interference. In other embodiments, the second dimension is a C18 column, such as a reverse phase column.The resolved steroid analytes may then be further analyzed, for example, by mass spectrometry.

[0076] Mass spectrometry analysis

[0077] The analytes separated by the current novel 2D LC system can be further analyzed by selected techniques. In most cases, mass spectrometry (MS) is used due to its ultra-high sensitivity. A mass spectrometer ionizes the target analyte, separates the resulting ions in a vacuum based on their mass-to-charge ratio, and finally measures the intensity of each ion. MS is very suitable for qualitative and quantitative analysis because the mass spectrum can indicate the concentration level of ions with a given mass.

[0078] A mass spectrometer consists of three main components: an ion source for ionization of the analyte, a mass analyzer for separating ions based on their mass-to-charge (m / z) ratio, and a detector for detecting the separated ions. In the ionizer, the 2D effluent is sprayed, desolvated, and ionized to produce charged particle ions. The ions migrate through a series of mass analyzers under vacuum. Precursor ions with a specific (m / z) ratio are selected to pass through the mass analyzer, excluding particles of all other (m / z) ratios. The separated ions are then detected, for example, by an electron multiplier. The ionization of the sample can be performed, 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, thermal spray / plasma spray ionization, or particle beam ionization. Ions can be detected by, for example, multiple reaction monitoring (MRM), selected ion monitoring mode (SIM), selected reaction monitoring (SRM), or an electron multiplier.

[0079] The MS data is transmitted to a computer, which plots the voltage versus time. 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 an internal standard to the test sample.

[0080] In some embodiments of the present disclosure, mass spectrometry methods utilize "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. The selection-fragmentation-detection sequence can be further extended to the first generation of product ions. For example, the selected product ions can be further fragmented to produce another set of product ions, and so on. The fragmentation pattern of the ions is highly specific to the structure of the compound and therefore allows accurate structural determination.

[0081] In some embodiments, the mass analyzer can 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).

[0082] The 2D LC-MS system of the present disclosure further comprises an interface for transferring the purified target analyte from the 2D column to the mass spectrometer. This interface is used due to the inherent incompatibility of liquid chromatography and mass spectrometry. Although the mobile phase solvent system in the LC system is a pressurized fluid, the MS device is usually operated under vacuum. The 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 and maintaining the chemical consistency of the target analyte.

[0083] In some embodiments, the interface is an electrospray interface. Alternatively, the interface can be an atmospheric pressure ionization interface, an atmospheric pressure chemical ionization interface, a thermospray interface, a moving belt interface, a guided liquid introduction interface, a particle beam interface, or an interface based on fast atom bombardment (FAB).

[0084] 2D LC-MS combines the excellent separation power of liquid chromatography with the excellent sensitivity and selective mass analysis capabilities of mass spectrometry to provide molecular mass and structural information of components in a mixture. This information can be supplemented by information derived from other LC detectors, including but not limited to refractive index detectors, chiral detectors, radio mobility detectors, UV detectors, fluorescence detectors, light scattering detectors, and conductivity detectors.

[0085] application

[0086] The novel 2D LC-MS systems (including novel 2D LC-MS / MS systems) of the present disclosure can be used to analyze (including detection and quantification) a variety of molecules, including small molecules (eg, drug substances) and large molecules (eg, biomolecules).

[0087] In some embodiments, the current 2D LC-MS / MS systems are used to monitor biomarkers in preclinical and clinical research and development for screening / diagnosis and therapeutic purposes. By way of example, the 2D LC-MS / MS systems can be used to measure the levels of certain lipid biomarkers in lysosomal storage diseases such as Fabry Disease, Gaucher Disease, Krabbe Disease, and acid sphingomyelinase deficiency (ASMD; e.g., Niemann-Pick Disease, type A, type B, and type A / B; NPD).

[0088] As further described in the working examples below, a 2D LC-MS / MS system can be used to analyze two lipid biomarkers in blood samples from ASMD patients: ceramide (CER) and lyso-sphingomyelin (lyso-SPM). ASMD is a disorder of sphingolipid metabolism that causes sphingomyelin to accumulate in tissues throughout the body, specifically the spleen, liver, lungs, bone marrow, and in some cases, the brain. Elevated levels of lyso-SPM (sphingosine phosphorylcholine) are also common. The deficient enzyme in patients (acid sphingomyelinase) catalyzes the hydrolytic fragmentation of sphingomyelin in lysosomes, thereby generating phosphorylcholine and ceramide. Because lipids such as ceramide and lyso-SPM are highly elevated in ASMD patients, they can be used as biomarkers to screen and diagnose ASMD and monitor enzyme replacement therapy (replacement with recombinant human ASM, such as olipudase alfa).

[0089] In order to better understand the present invention, the following examples are described. These examples are for illustrative purposes only and should not be considered to limit the scope of the present invention in any way. Example

[0090] Example 1: 2D LC-MS / MS Detection of Ceramide and Hemolytic SPM in Human Plasma

[0091] This example describes the use of the novel 2D LC-MS / MS system of the present disclosure to analyze ceramide (CER) and hemolytic SPM in human plasma samples.

[0092] Chemicals and reagents

[0093] 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 (sphingosine phosphatidylcholine) were purchased from Matreya (PA, USA). Lyso-SPM internal standard (d9-lyso-sphingomyelin) was synthesized in-house. Methanol and acetonitrile (both HPLC grade) were purchased from Honeywell (NC, USA). Ammonium acetate was purchased from Sigma Aldrich (MO, USA). Deionized water was obtained using an in-house MilliQ DI system (Millipore, MA, USA). Formic acid (Optima TM LC / MS grade) was purchased from Fisher Scientific (Hampton, NH). Trifluoroacetic acid (TFA) was purchased from EMD Millipore (MA, USA).

[0094] Lipid extraction

[0095] 10 μL of human plasma was mixed with 800 μL of analytical working solution (80% methanol, 20% acetonitrile, 10 mM ammonium acetate and 1% formic acid) containing the internal standard (IS) of CER (0.22 μg / mL) in a 96-well culture plate. After vortexing for 10 min on a DVX-2500 multitube vortexer (VWR Scientific Products, NJ, USA) at 11,000 rpm and sonicating for 10 min in a water bath (Branson 3510 ultrasonic cleaner, Marshall Scientific, NH, USA), the culture plate was subjected to 5 min centrifugation in a reverse centrifuge (Beckman Coulter, CA, USA). The supernatant was transferred to a new 96-well culture plate and provided for 2DLC-MS / MS analysis. Based on the standard curve using a linear fit, the CER concentration in each sample was quantified as μg / mL in plasma and the hemolytic SPM was quantified as ng / mL.

[0096] LC-MS / MS system settings

[0097] 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). Software (AB Sciex) control. Use Microsoft Excel and Watson LIMS TM Data processing and analysis were performed using the HER2 software (Thermo Fisher Scientific, Waltham, MA).

[0098] The 2D LC setup consisted of 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. The two HPLC columns were placed inside the Acquity column manager so that each column occupied a different compartment within the column manager and the two columns were kept at the same temperature (60° C.). The 2D HPLC setup included the following connections:

[0099] a) PEEK sleeve (about 60 cm in length) connecting the outlet of the first dimension analytical column to port 1 of the diverter valve;

[0100] b) a second PEEK sleeve (about 70 cm in length) connecting port 2 of the diverter valve to the inlet of the second dimensional analytical column;

[0101] c) a third PEEK sleeve (about 80 cm in length) connecting the outlet of the second dimension analytical column to the mass spectrometer; and

[0102] d) A fourth PEEK sleeve (at least 60 cm in length) connecting port 3 of the diverter valve to waste.

[0103] Mobile phase

[0104] Mobile phase A was water plus 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 into the LC system at predetermined time points of the run, as indicated in Table 1 below. The injection volume of the sample was 2 μL injection, and the mobile phase flow rate was 0.2 mL / min.

[0105] Table 1.2D LC solvent conditions

[0106] Time point (min) A％ B％ 0.0 15 85 2.00 15 85 2.20 30 70 4.00 30 70 4.20 1 99 17.50 1 99 17.60 15 85 20.00 15 85

[0107] LC Diverter Valve Conditions

[0108] In conjunction with the adjustments of the solvent system as indicated in the table above, the diverter valves of the 2D LC system were switched to different port positions at predetermined time points as indicated in Table 2 below to allow different portions of the ID effluent to be directed to waste or the next (RP) column. Figure 4 .

[0109] Table 2.2D LC-MS / MS diverter valve conditions

[0110]

[0111] Mass spectrometry analysis

[0112] Multiple reaction monitoring (MRM) techniques were used to analyze ceramide and lytic SPM. This technique uses a triple quadrupole MS that first targets ions corresponding to the compounds of interest, ceramide and lytic SPM, and then fragments the target ions to produce a series of daughter ions. One (or more) of these fragmented daughter ions is selected for quantitative purposes. Only compounds that meet these two criteria, i.e., a specific parent ion and a specific daughter ion corresponding to the mass of the compound, are separated within the mass spectrometer.

[0113] The MRM transition parameters in the API 4000 mass spectrometer are shown in Table 3 below.

[0114] Table 3. Ceramide MRM transitions

[0115]

[0116] Table 4 shows the MRM transition parameters for the ceramide internal standard (IS), the hemolytic SPM analyte, and the hemolytic SPM IS.

[0117] Table 4. Ceramide IS, Lyso-SPM, and Lyso-SPM IS MRM Transitions

[0118]

[0119] result

[0120] In this novel analytical method, CER and lyso-SPM are simultaneously extracted with an analytical working solution compatible with the mobile phase solvent system subsequently used in the 2D LC system. The extracted sample is then directly injected into the first column and transferred to the second column by continuous flow without the need for solvent exchange. We have found that normal phase chromatography is well suited for separating CER and lyso-SPM from phospholipids. Therefore, this method is used as the first dimension of LC separation. The second dimension of separation using reversed phase chromatography is used to separate CER and lyso-SPM from structurally related compounds (such as glycosphingolipids and lyso-glycosphingolipids).

[0121] More specifically, in this LC process, the diverter valve is set at position A at the beginning to allow the column to be flushed. The valve is then set at position B, and the ceramide separated from the phospholipids in the normal phase (NP) column is allowed to flow to the RP column and be retained in the RP column. The diverter valve is then switched to waste (position A) to eliminate the phospholipids and other matrix components that follow in the ID effluent. Finally, the diverter valve is switched again to position B to allow the lytic SPM to enter the RP column, where both ceramide and lytic SPM are separated from other potential interfering molecules prior to mass spectrometry analysis.

[0122] The data showed that CER and hemolytic SPM were successfully extracted and quantified. Figure 4As shown in , ceramide elutes first from the NP column, followed by the main impurity phospholipids, and then the lyso-SPM. The phospholipids are directed to waste by setting the diverter valve to position A, where the ceramide and lyso-SPM analytes are directed to the RP column by setting the valve at position B. In the RP column, the lyso-SPM appears as a single peak, while the ceramide analyte is divided into different retention windows containing the following isoforms according to the fatty acid chain length and the number of double bonds: C16, C18, C20 and C22:1, C22 and C24:1, C24, and C26:1. The data show that the CER isoforms eluted from the RP column are sharp peaks with almost no background noise, and a single peak for each isoform is identified. Several runs were performed and they gave consistent retention times.

[0123] The above 2D LC-MS / MS method reduces the sample turnaround time for quantification of CER and hemolytic SPM in human plasma from the usual 8 days to only 2 days. In addition, the original labor-intensive offline extraction work for sample cleanup, including SPE and protein precipitation (PPT) procedures, was reduced from 25 hours to 1 hour by utilizing current extraction solvents and continuous flow 2D LC. See, e.g., Chuang et al., Methods in Molecular Biology, Vol. 1378, DOI 10.1007 / 978-1-4939-3182-8_28. As can be seen above, the entire 2D LC run was completed in 20 minutes.

[0124] Table 5. Comparison between 1D LC-MS / MS and novel 2D-LC-MS / MS

[0125]

[0126] Example 2: Analytical Verification of 2D LC-MS / MS Detection of Ceramide in Human Plasma

[0127] This example describes a study to validate a 2D LC-MS / MS system for the detection of ceramide. In this study, samples, calibration curve standards, and controls were added to a 96-well plate. An analytical working solution containing a known amount of an internal standard (CER-IS) was then added to the wells. The plate was then vortexed, sonicated, and centrifuged as described in Example 1. After centrifugation, the supernatant was transferred to a new 96-well plate. The new plate was then transferred to the 2D LC-MS / MS system for analysis, also as described in Example 1.

[0128] After the sample is run, use Analyst TMThe software integrates the peaks corresponding to the individual CER isoforms. The peak areas of the individual CER isoforms for any given sample are then summed in Excel. The total CER peak area is imported into It calculated total CER concentration (μg / mL) based on the ratio of total CER peak area to CER-IS peak area using the calibration curve intercept and slope.

[0129] The novel multi-task assay was validated for CER quantification. Parameters validated included system suitability, intra-run and inter-run accuracy and precision, calibration (standard) curve linearity, analytical sensitivity, carryover, delipidated plasma matrix effects, reinjection reproducibility, batch size assessment, extract recovery, sample dilution integrity, determination of CER reference in healthy donors, and comparison of methods for determining CER content in plasma.

[0130] Carryover was assessed by injecting solvent (only working solution was analyzed) after reaching the highest calibration standard in each run. Delipidated plasma matrix effects were assessed using three independent batches of delipidated plasma spiked with three different concentrations of analyte (low quality control or LQC, medium quality control or MQC, and high quality control or HQC). Reinjection reproducibility was assessed by continuing to re-inject samples at LLOQ (lower limit of quantification), LQC, MQC, HQC, and ULOQ (upper limit of quantification) concentrations. Extraction recovery was assessed by comparing 2 combined patient samples before and after spiking with CER. An internal standard (IS) was introduced at the same stage for the conditions before and after spiking.

[0131] The data show that the current novel 2D LC-MS / MS method detected 11 different CER isoforms by multiple reaction monitoring (MRM) using independent MS / MS channels for each of these isoforms. System suitability testing showed that the CV% was between 0% and 5% in all run-throughs performed throughout the validation process. The mean %Bias and mean %CV (logarithmic change) for all concentrations passed the established accuracy and precision criteria. Analytical sensitivity was defined as the lowest concentration of an analyte that could be measured with acceptable accuracy and precision. The CV% and mean %Bias for LLOQ were 8% and -8%, respectively.

[0132] The minimum and maximum carryover of the analyte peak area were 2% and 11%, respectively. The overall CV% of the matrix effect of delipidated plasma measured at three concentrations was 6% (LQC), 2% (MQC), and 3% (HQC). For the specified concentrations, the CV% of the reinjection reproducibility ranged from 2% to 4%. The extraction recovery ranged from 67% to 88%, with the CV% varying from 2% to 11%. The results confirmed that the method is precise and accurate and has been validated for measuring ceramide concentrations in human plasma.

Claims

1. A method for monitoring one or more lipid biomarkers in a blood sample of a subject in need thereof, the method comprising the steps of: (a) extracting the one or more lipid biomarkers from the blood sample using an extraction solvent to obtain an extracted sample; (b) applying the extracted sample and solvent system to a first liquid chromatography (LC) column using an LC pump system, wherein the first LC column is directly connected to a second LC column via a cannula having a diverter valve; (c) setting the diverter 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 diverter 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 a sample obtained from the chromatographic separation of the second LC column, The method described therein uses only one LC pump system for both LC columns.

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

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

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

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

8. A method according to any one of the preceding claims, wherein the relative ratios of the components of the solvent system are varied during a single sample run.

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

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

11. The method of claim 9 or 10, wherein the subject is being treated with or is in need of enzyme replacement therapy.

12. The method of claim 10, wherein the lysosomal storage disease is ASMD, optionally wherein the one or more lipid biomarkers comprise ceramide and / or lyso-sphingomyelin.

13. The method of claim 12, wherein the one or more lipid biomarkers 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 of any one of claims 9-13, wherein the first LC column is a silica column and the second LC column is a C18 column.

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

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

17. The method of claim 16, wherein the solvent system is obtained by mixing a first solvent comprising water and 0.5% trifluoroacetic acid and a second solvent comprising 85% methanol (v / v), 15% acetonitrile (v / v) and 0.5% trifluoroacetic acid, optionally wherein the ratio of the first solvent to the second solvent is 70:30, 85:15 or 99:1.

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