Improved lipid extraction

Abstract

This document provides workflows for separating metabolites and lipids (“2in1”) or separating metabolites, lipids, and proteins (“3in1”). The 2in1 method involves: treating a sample containing metabolites and lipids with a mixture of methanol and ethanol to precipitate proteins; adding water to improve metabolite recovery; and passing the treated sample through a lipid-affinity solid-phase extraction (SPE) matrix. The effluent from the SPE matrix contains metabolites, and lipids can be eluted from the SPE matrix using a lipid elution solution. The 3in1 method is similar to the 2in1 method, except that proteins are granulated prior to SPE extraction, and then the granules are analyzed for protein content.

Description

[0001] Cross-references to related applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 548,338, filed November 13, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure particularly provides a method for extracting lipids from a sample. The method includes the steps of: precipitating proteins and recovering lipids from the supernatant, washing protein particles, and then collecting lipids from the wash solution. In some embodiments, the method improves the recovery of certain lipid classes, such as triglycerides (TG, or sometimes referred to as TAG) and cholesterol esters (CE). The method may optionally further include extracting metabolites, proteins, and / or nucleic acids. After extraction, the extracted analytes can be analyzed, for example, using mass spectrometry or other methods known in the art. Summary of the Invention

[0003] This article provides a method for extracting lipids from a sample, the method comprising: (a) Precipitate proteins from the sample to produce a sample mixture comprising protein precipitate and supernatant, and separate the protein precipitate from the supernatant; (b) Passing the supernatant through a solid-phase extraction matrix with an affinity for lipids to produce a first effluent; (c) Wash the protein precipitate with a first lipid elution solution to obtain a washing solution; (d) Elute the solid-phase extraction matrix with a second lipid elution solution after (b) to obtain a second effluent; The washing liquid and the second effluent contain lipids.

[0004] The method may further include adding water to the supernatant before passing the supernatant through the solid-phase extraction matrix. A second lipid elution solution may contain the washing solution. The first and second lipid elution solutions may have the same composition. The method may further include additional elution of the solid-phase extraction matrix with the lipid elution solution after step (d). The method may further include collecting metabolites from the first effluent.

[0005] The method may further include: eluting the solid-phase extraction matrix with a metabolite elution solution prior to (d) to collect the metabolites in the resulting eluent. The method may further include: combining the resulting eluent with the first effluent.

[0006] The method may further include collecting the protein from the protein precipitate. In this method, the protein precipitate may not be separated from the supernatant by filtration. The protein precipitate may be separated from the supernatant by centrifugation. The protein may be precipitated using a solution containing methanol and ethanol. The protein may be precipitated using a 50:50 methanol / ethanol (v / v) solution. During the separation, the water content of the sample mixture is 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less.

[0007] The sample can be plasma, serum, body fluids other than serum or plasma, or lysed cells. Cells can be derived from blood, cultured suspension cells, cultured adherent cells, 3D cultured cells (e.g., organoids, spheroids, cell cultures grown in 3D supports), or tissues.

[0008] The sample may contain cells that have been treated with a solution containing a fluorinated alcohol. The fluorinated alcohol may contain one or more of the following: 2,2,2-trifluoroethanol, 2,2-difluoroethanol, 2-fluoroethanol, hexafluoro-2-propanol, nonafluoro-tert-butanol, 1,1,2,2,2-pentafluoroethanol, and / or 2,2,3,3,3-pentafluoro-1-propanol. The fluorinated alcohol may be 2,2,2-trifluoroethanol.

[0009] The method may include automated steps.

[0010] At least one of the lipid elution solutions can be 2:1 methanol:dichloromethane.

[0011] Other objects, features, and advantages of the invention will become apparent from the following detailed description (or “details”). However, it should be understood that while the detailed description and specific embodiments indicate preferred embodiments of the invention, they are given by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art based on this detailed description. Attached Figure Description

[0012] The following figures form part of this specification and are included to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these figures in conjunction with the detailed description of specific embodiments given herein.

[0013] Figure 1 Mass spectra showing lipid recovery profiles after the 2in1 (black) and 3in1 (gray) workflows.

[0014] Figure 2Mass spectra showing the lipid recovery characteristics after 2in1 (black), 3in1 (no particle washing, light gray), and the new 3in1 (with particle washing, dark gray) workflows.

[0015] Figure 3 Recovery rates of various lipid classes when using 2in1 (light gray), 3in1 (particle-free washing, dark gray), and the new 3in1 (particle washing, gray) workflows.

[0016] Figure 4 A block diagram of one embodiment of the unified workflow 10 of this disclosure. Detailed Implementation

[0017] definition In this disclosure, the term "metabolite" refers to one or more compounds that serve as substrates and / or products of metabolic processes (reactions). Metabolites are typically small molecules (less than 1500 Da) rather than proteins or nucleic acids, although amino acids, small peptides (e.g., dipeptides or tripeptides), nucleotides, and small oligonucleotides (e.g., dinucleotides or trinucleotides) can be metabolites. Metabolites can include substrates and / or products produced by metabolic processes (reactions) in living cells, including but not limited to reactions of central carbon metabolism, including those involved in glycolysis, the tricarboxylic acid cycle (i.e., the TCA cycle, Krebs cycle), the reduced pentose phosphate cycle (i.e., the Calvin cycle), glycogen metabolism, the pentose phosphate pathway, and other metabolic processes. Therefore, metabolites may include, but are not limited to, glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, 1,3-diphosphoglyceric acid, 3-phosphoglyceric acid, 2-phosphoglyceric acid, phosphoenolpyruvate, pyruvate / ester, acetyl-CoA, citrate, cis-aconitate (salt / ester), d-isocitric acid (salt / ester), α-ketoglutarate (salt / ester), succinyl-CoA, succinic acid (salt / ester), and rich in other metabolites. Horse acid (salt / ester), malic acid (salt / ester), oxaloacetic acid (salt / ester), ribulose-1,5-bisphosphate, 3-phosphoglyceric acid, 1,3-bisphosphoglyceric acid, glyceraldehyde-3-phosphate, ribulose-5-phosphate, ethanol, acetaldehyde, pyruvate, 6-phosphogluconolactone, 6-phosphogluconolactone, 6-phosphogluconolactone, 6-phosphogluconolactone, ribose-5-phosphate, xylulose-5-phosphate, sedoheptulose-7-phosphate, erythrose-4-phosphate and other metabolites.

[0018] Metabolites include compounds of various chemical classes and having various chemical structures, including but not limited to organic acids, sugars, sugar phosphates (salts / esters), amino acids, nucleobases, nucleotides, drug metabolites, steroids, fatty acids, and triglycerides. Some metabolites are lipids or lipophilic compounds, including lipids modified with one or more polar groups during various metabolic reactions. These metabolites may be referred to herein as lipids, polar lipids, or lipid metabolites.

[0019] Metabolites may include pharmaceutical compounds and pharmaceutical metabolites, or food compounds and food metabolites. Therefore, metabolites in this disclosure include both metabolites naturally produced by cells and / or organisms, and / or exogenous substances.

[0020] Exogenous substances are chemical compounds (such as antibiotics, inactivated steroids, or any drug metabolites) that are present in a biological sample (such as urea or blood) but are not naturally occurring or are not expected to be present in that biological sample, or are not expected to be present in the amount present in the biological sample.

[0021] The term "metabolic reaction" refers to any chemical reaction involved in catabolism and / or anabolism. These are any chemical reactions that occur in living organisms, typically within living cells (in vivo or in vitro).

[0022] The term "metabolism" is the sum of all metabolic reactions.

[0023] The term "biological sample" refers to a whole organism or a subset of its tissues, cells, and / or components (e.g., tissue and cell cultures, body fluids, including but not limited to blood, plasma, serum, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, gastric juice, amniotic fluid, amniotic fluid, cord blood, urine, vaginal fluid, semen, and feces). "Biological sample" can also refer to whole cells, homogenates, lysates, or extracts prepared from a whole organism or a subset, fraction, or part thereof, including but not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, external portions of the skin, respiratory tract, intestine, and genitourinary tract, tears, saliva, breast milk, blood cells, tumors, and organs. In some embodiments, the biological sample is derived from animals, plants, and / or fungi.

[0024] The biological samples disclosed herein may comprise cells. The term "cell" is used in its conventional sense to refer to the basic structural unit of a living eukaryotic or prokaryotic organism. In some embodiments, cells include prokaryotic cells, such as bacteria or archaea. Cells may include eukaryotic cells. Cells may include plant cells or fungal cells. Cells may include, but are not limited to, cultured cells (e.g., tissue culture cell lines), bacterial cells, recombinant cells (which are cells that have been modified in a laboratory by, for example, gene editing or by any other means known to those skilled in the art), yeast cells, and / or primary cells that may be obtained from animals, humans, plants, and / or fungi. Biological samples may comprise fungal cells, cells of the archaea domain, or cells comprising a larger biological sample (e.g., cells from a biopsy or tissue sample). Cultured cells include, but are not limited to, cells in suspension culture, cells harvested from adherent cells, or cells from 3D cultures (e.g., organoids, spheroids, cell cultures grown in 3D supports).

[0025] In some methods of the present invention, a solution containing a fluorinated alcohol is used to lyse a biological sample containing cells. “Cell lysis” means that the cell membrane is at least partially permeable, allowing at least some cellular contents (including at least some metabolites) to leak out of the cell. In some cases, the cell membrane may rupture or break open. In some embodiments, cell lysis may further include the lysis of organelles (e.g., the nucleus, mitochondria, ribosomes, chloroplasts, lysosomes, vacuoles, Golgi apparatus), allowing the contents of the organelles to also be released into the surrounding medium.

[0026] In this disclosure, the term "fluoroalcohol" refers to an organofluorine compound comprising a hydroxyl group (-OH) and one or more fluorine-carbon bonds. Preferably, a fluoroalcohol is an organofluorine compound comprising a hydroxyl group (-OH) and one or more of a fluoroalkyl, fluoroalkenyl, and / or fluoroalkynyl group. Suitable fluoroalcohols include those having a terminal and / or internal fluoroalkyl, fluoroalkenyl, and / or fluoroalkynyl group. A more detailed description of fluoroalcohols can be found, for example, in U.S. Patent Application Publication No. 20200393342, the entire contents of which are incorporated herein by reference, particularly with respect to the various methods and reagents disclosed therein for the separation of metabolites, lipids, proteins, and / or nucleic acids.

[0027] Description of the invention The inventors have developed workflows for separating metabolites and lipids (“2in1”) or separating metabolites, lipids, and proteins (“3in1”). The 2in1 method includes: treating a sample containing metabolites and lipids with a mixture of methanol and ethanol to precipitate proteins; adding water to improve metabolite recovery; and passing the treated sample through a lipid-affinity solid-phase extraction (SPE) matrix. The effluent from the SPE matrix contains metabolites, and lipids can be eluted from the SPE matrix using a lipid elution solution. The 3in1 method is similar to the 2in1 method, except that the proteins are granulated prior to SPE extraction. Protein analysis is then performed on the granules.

[0028] We unexpectedly noticed that lipid recovery was affected in the 3in1 workflow. Lipids recovered from plasma samples using both the 3in1 and 2in1 methods were analyzed by mass spectrometry. Figure 1 Comparing them with each other.

[0029] like Figure 1 As shown, certain lipid classes were selectively reduced in the 3in1 results, particularly the lipids eluted from the LC column later in the triglyceride and cholesterol ester elution regions.

[0030] This invention improves lipid recovery by washing protein particles in a 3-in-1 method with a lipid elution solution and by recovering lipids from the washing solution in addition to the lipids obtained from eluting the SPE matrix. Figure 2 The results of the improved 3-in-1 and 2-in-1 workflows are shown, and the lipid recovery rates are generally comparable. In fact, the improved 3-in-1 workflow unexpectedly achieved even higher recovery rates for some lipid classes (such as triglycerides and cholesterol esters) than the 2-in-1 workflow.

[0031] In the following Figure 3 The bar chart in the image more clearly shows the differences in recovery rates for various lipid categories.

[0032] Therefore, the present invention provides a method for extracting lipids from a sample, comprising: precipitating proteins from the sample and separating the protein precipitate from a supernatant; washing the protein precipitate with a first lipid elution solution to obtain a washing solution; passing the supernatant separated from the protein precipitate through a solid-phase extraction matrix having an affinity for lipids to produce a first effluent; and eluting the solid-phase extraction matrix with a second lipid elution solution to obtain a second effluent, wherein both the washing solution and the second effluent contain lipids. Optionally, the solid-phase extraction matrix can be further eluted with an additional lipid elution solution to collect additional lipids in the eluent. The washing solution and the second effluent (and any other additional eluents) can be combined for lipid analysis and / or recovery. In fact, in some embodiments, the washing solution is used to elute the solid-phase extraction matrix (i.e., the second lipid elution solution is or contains the washing solution). In some other embodiments, the first lipid elution solution and the second lipid elution solution are the same. In other embodiments, the second lipid elution solution does not contain the washing solution and is compositionally different from the first lipid elution solution. Lipids can be analyzed and / or collected for further use by any method known in the art. The lipid elution solution can be any solution suitable for the specific solid-phase extraction matrix used, as can be determined by a person of ordinary skill in the art. For example, the lipid elution solution may contain dichloromethane and / or chloroform, and optionally also contain one or more of MTBE, butanol, methanol, ethanol, and isopropanol. In some embodiments, the lipid elution solution is a 2:1 methanol:dichloromethane.

[0033] The first effluent contains polar metabolites. Optionally, after protein removal and before passing through the solid-phase extraction matrix, the supernatant can be diluted with water to improve the recovery of metabolites in the first effluent. Optionally, after collecting the first effluent, the solid-phase extraction matrix can be eluted with a metabolite elution solution, and the effluent can be combined with the first effluent to collect the metabolites.

[0034] In some embodiments, an organic solvent is used to precipitate proteins from the sample, such as a solution containing methanol and ethanol (e.g., a 50:50 (v / v) methanol:ethanol). For example, 20 μL of plasma can be mixed with 112.5 μL of a 1:1 methanol:ethanol solution, and the resulting protein precipitate can be separated from the supernatant. In this case, the water content of the sample mixture is 15% when the protein precipitate and supernatant are separated. If water is added to the sample mixture after precipitation but before the protein precipitate is separated, the water content of the sample mixture will be higher than 15%. In some embodiments, the water content at this separation is at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, or at most 5%. As mentioned above, in some embodiments, water is added to the supernatant after the protein precipitate is removed to increase the water content in the supernatant. The protein precipitation solvent can be any solvent or combination of solvents known in the art, such as ethanol:acetonitrile (e.g., 1:1), ethanol:methanol (e.g., 1:1), or ethanol.

[0035] The sample may be a biological sample. In some embodiments, the sample contains cells. In some embodiments, the sample (especially a sample containing cells) is first treated with a fluorinated alcohol to lyse the cells and / or stop the metabolic reaction (i.e., quench metabolism), as described in more detail in U.S. Patent Application Publication No. 20200393342.

[0036] In some embodiments, the present invention is used to detect and analyze lipid characteristics, which can aid in the diagnosis, monitoring, and treatment of, for example, lipid disorders. Lipid disorders include, but are not limited to, primary lipid disorders such as hypercholesterolemia and hypertriglyceridemia, and disorders that cause abnormal lipid characteristics, such as diabetes, hypothyroidism, nephrotic syndrome, nephropathy, obstructive liver disease, and abnormal proteinemia. Similarly, lipid characteristics can also be used to monitor the effects of drugs that alter lipid characteristics.

[0037] In some embodiments, in addition to lipids, other analytes, such as metabolites, proteins, and / or nucleic acids, are detected and analyzed. In some embodiments, proteins in protein precipitates obtained from samples are analyzed. In some embodiments, the first effluent from the SPE matrix contains metabolites and can be analyzed accordingly. In some embodiments, nucleic acids are separated (e.g., by filtration) before protein precipitation. Any combination of analytes can be selected according to the user's purpose, such as lipids and metabolites, lipids and proteins, lipids and nucleic acids, and lipids, metabolites, and proteins.

[0038] Sample 16 can be a fluid, such as plasma or serum, which can be directly introduced into box 22. Alternatively, the sample may initially contain cells, such as in box 12. The option of using a sample containing cells is depicted by dashed lines. The sample containing cells is contacted with a metabolic quenching (MQ) solution, as shown in box 14. This results in cell lysis and metabolic quenching, producing a mixture in box 16 containing one or more metabolites and proteins / peptides, lipids, and / or nucleic acids.

[0039] Metabolites in the mixture are protected from becoming substrates for metabolic reactions. "Protected" means that at least some enzymes in the mixture are at least partially inactivated and at least some metabolic reactions are inhibited. Therefore, at least some metabolites are at least partially protected (quenched) from becoming substrates for metabolic reactions for at least a period of time.

[0040] Biological samples can be prepared prior to contact with the MQ solution. For example, cells can be granulated (e.g., by centrifugation) and optionally washed and / or resuspended in a suitable buffer. For example, cells can be filtered through a filter plate to remove cell culture medium and optionally washed with a suitable buffer. For example, cell culture medium can be removed from adherent cells, and a washing buffer, such as PBS or isotonic ammonium bicarbonate, can be added to remove it ( wholly or partially) from the cells.

[0041] Optionally, the sample from box 16 can be filtered at box 18, or this step can be omitted. Suitable filtration methods may employ membrane filters, PVDF (polyvinylidene fluoride), nylon, PTFE (polytetrafluoroethylene), PC (polycarbonate), PP (polypropylene), PES (polyether sulfone), PVC (polyvinyl chloride), CA (cellulose acetate), CMF (coated cellulose acetate), HDPE (high-density polyethylene), regenerated cellulose and / or glass fiber filters, or stacked combinations of filter types with appropriate pore sizes suitable for the sample used. Other types of filters may also be used. In some applications, the mixture is filtered through a glass fiber filter at box 18. Various glass fiber filters typically used for separating nucleic acids can be used. For example, borosilicate glass fiber filters with various pore sizes can be used. Typically, pore sizes can range from 0.5 μm to 3 μm. The pore size is within the μm range. The pore size can be adjusted to optimize recovery from specific biological samples. For trace samples, filtration can be performed in a filter plate, such as a 96-well or 384-well plate. Glass fiber filter plates are commercially available from many different suppliers.

[0042] The filtration process can be further optimized as needed, and may include applying vacuum pressure and / or positive pressure. The filtration may also include a washing step, in which the filter is washed using an additional fluorinated alcohol solution or other solution. This can increase the recovery of metabolites or other components from the lysate.

[0043] The pore size of the glass fiber filter can be further optimized, depending on whether nucleic acids, such as DNA and / or RNA, need to be analyzed. In some embodiments, an uncoated glass fiber filter is used. In other embodiments, the glass fiber filter can be coated with an adhesive to improve the binding of nucleic acids (NAs) to the filter. Additives or adhesives can also be used to repel the binding of certain metabolites or other cellular components.

[0044] Nucleic acids (e.g., DNA and / or RNA) collected at box 18 on a glass filter or otherwise can be removed and collected at box 20. One method for removing DNA from the glass filter is to add a solution with a low (acidic) pH to box 20 to protonate the DNA and disrupt some of the bonding interactions between the DNA and the glass. Another commonly used method is to elute DNA and / or RNA with a low-salt buffer. A low-salt buffer can be prepared using a high concentration (approximately 1M) of EDTA. Another low-salt buffer is purified deionized water with salt ions removed.

[0045] The filter at box 18 can be set so that other components of the lysed biological sample (proteins, lipids, and metabolites) can flow through while nucleic acids are captured.

[0046] Following filtration at box 18, or, if filtration is omitted, proceeding directly from box 16, protein precipitation of the sample or effluent lysate solution containing proteins, lipids, and metabolites is performed at box 22. These protein precipitates are formed into particles, and proteins and peptides can be collected from the particles for further use (box 24). The supernatant is passed through the SPE matrix (box 26), and the particles are washed with a lipid elution solution (box 36). In some embodiments, the washing solution is used to elute the SPE matrix as described above in this disclosure. Figure 4 As shown in the diagram. Alternatively, the SPE matrix can be eluted with a lipid elution solution without washing buffer to obtain an eluent. Both the eluent and the washing buffer contain lipids and can be combined or kept separate for further analysis and / or lipid recovery.

[0047] Various SPE matrices are known. Matrices that separate lipids from metabolites based at least in part on the affinity of lipids for the matrix are preferred matrices in the methods of the present invention because they allow for robust separation of polar metabolites from lipids. Suitable SPE matrices include solid-phase adsorbents described in U.S. Patent Publication 2018 / 0080858. Commercially available SPE columns and plates can also be used. One of the commercially available product families for capturing lipids and separating metabolites from lipids is CAPTIVA, which is commercially available from Agilent Technologies, Santa Clara, California, USA. TM EMR-lipid SPE columns and plates. Other commercially available adsorbents can also be used.

[0048] In box 26, metabolites flow through an EMR-lipid column or plate, and the adsorbent captures the lipids, providing a solution primarily containing polar metabolites, which can be used for metabolite analysis. It should be noted that under certain purification conditions, some lipid compounds (e.g., short-chain fatty acids, sterols, etc.) can be found in the solution containing metabolites (including polar metabolites).

[0049] After collecting lipids in the effluent solution at box 28, they can be analyzed by liquid chromatography / mass spectrometry (LC / MS) at box 40. The solvent can be changed before analysis at box 40 if needed. This also applies to metabolite fractions of the sample, and also to nucleic acid fractions. Solvent changing also provides the opportunity to make the fraction more concentrated, thus making low-abundance analytes easier to detect by LC / MS.

[0050] LC / MS and other analytical methods can also be used for lipids, metabolites, proteins / peptides, and nucleic acids. Figure 4 The schematic diagram illustrates LC / MS analysis. However, compounds obtained using the methods of this invention can be analyzed by any other methods commonly used in the art.

[0051] Example 1 Lipid identification using various workflows As described above, the plasma samples were subjected to 2in1, 3in1 (particle-free washing), and a new 3in1 (precipitate washing) workflows, with the following results: Figures 1-3 As shown. The materials and methods are described below.

[0052] 2-in-1 (Metabolites and Lipids) Workflow In short, plasma samples were mixed with a 1:1 methanol:ethanol mixture and incubated, followed by the addition of water. The mixture was then passed through a Captiva EMR-LipidLipid plate (Agilent Technologies). Eluent containing polar metabolites was collected. The plate was eluted with two 2:1:1 water:methanol:ethanol solutions to collect more metabolites from the elution. The plate was then eluted with a lipid elution solution (2:1 methanol:dichloromethane). Lipids were collected in the eluent. The plate was eluted again with the same lipid elution solution, and the eluent was combined with the first eluent, dried, reconstructed, and analyzed by mass spectrometry.

[0053] Automation can be added to a 2-in-1 workflow. For example: 1. Transfer 20 μL of plasma aliquots into the wells of a 96-well plate.

[0054] 2. Add approximately 130-160 μL of a 1:1 methanol:ethanol mixture to each well of a separate 96-well plate.

[0055] a. In some iterations, the 1:1 methanol:ethanol solution included a pre-spike of deuterium-labeled lipids (dissolved in 1:1 methanol:ethanol) to assess lipid recovery. The pre-spike of deuterium-labeled lipids could also be used as an internal standard.

[0056] b. As an alternative, a 1:1 methanol:ethanol ratio can be added to a single-well reservoir.

[0057] 3. Pour the water:methanol:ethanol ratio of 2:1:1 into the single-hole storage tank.

[0058] a. As an alternative, a 2:1:1 ratio of water:methanol:ethanol can be added to the wells of a 96-well plate.

[0059] 4. Add milliQ water to the 96-well plate and add 92.5 μL (112.5 μL minus the plasma sample volume) to the wells of the plate.

[0060] a. As an alternative, water can be poured into a single-hole reservoir.

[0061] 5. Add the plasma plate, 1:1 methanol:ethanol plate, water plate, and 2:1:1 water:methanol:ethanol plate to the Agilent Bravo Liquid Handler equipped with supplementary protocols for processing plasma samples. All other necessary consumables (e.g., Captiva EMR-Lipid plates, metabolite collection plates, and pipette tips for liquid and sample transfer) to the Bravo as well.

[0062] 6. Using the supplementary protocol, Bravo transferred 112.5 µL of a 1:1 methanol:ethanol solution to the plate containing the plasma sample. Mix the plasma and 1:1 methanol:ethanol solution using a pipette and plate shaking. The 1:1 methanol:ethanol solution precipitates proteins from the plasma.

[0063] 7. Complete the optional 10-minute incubation to equilibrate the sample.

[0064] a. Bath sonication is not recommended for these samples, as it will significantly slow down the filtration speed of the samples through the Captiva EMR-Lipid plate.

[0065] 8. Bravo transferred the plasma and 1:1 methanol:ethanol mixture into the wells of a plate containing 92.5 µL (112.5 µL minus the plasma sample volume) of water. The sample was mixed using a pipette.

[0066] a. Alternatively, Bravo can transfer 92.5 µL (112.5 µL minus the plasma sample volume) of water to each well containing a mixture of plasma and 1:1 methanol:ethanol. For this option, the sample can be mixed using pipette mixing and plate shaking.

[0067] 9. Complete the second optional 10-minute incubation to equilibrate the sample.

[0068] a. Bath sonication is not recommended for these samples, as it will significantly slow down the filtration speed of the samples through the Captiva EMR-Lipid plate.

[0069] 10. Transfer the sample (plasma + 1:1 methanol: ethanol + water) to a Captiva EMR-Lipid plate. Use vacuum pressure to pull the sample through the plate.

[0070] a. After initial filtration at a lower vacuum pressure, use a higher vacuum pressure to elute additional sample solution.

[0071] b. Alternatively, positive pressure can be used to push the sample through the plate. After initial filtration at a lower vacuum pressure, a higher positive pressure can be used to elute additional sample solution.

[0072] 11. Collect polar metabolites from the effluent.

[0073] 12. Wash the Captiva EMR-Lipid plate with 250 μL of a 2:1:1 water:methanol:ethanol solution, and then use vacuum pressure to move the solution through the plate. Collect the eluent along with the previous eluent and add the polar metabolite fraction.

[0074] a. Before adding 250 µL of 2:1:1 water:methanol:ethanol to the Captiva EMR-Lipid plate, wash the plate with 250 µL of 2:1:1 water:methanol:ethanol to the plate that previously contained a plasma + 1:1 methanol:ethanol + water sample.

[0075] b. After initial filtration at a lower vacuum pressure, use a higher vacuum pressure to elute additional wash solution.

[0076] c. Alternatively, a positive pressure can be used to move a 2:1:1 water:methanol:ethanol solution through the plate. After an initial filtration at a lower positive pressure, a higher positive pressure can be used to elute additional wash solution.

[0077] 13. Wash the Captiva EMR-Lipid plate a second time with 250 μL of a 2:1:1 water:methanol:ethanol solution, and then use vacuum pressure to move the solution through the plate again. Collect the eluent together with the previous eluent and add the polar metabolite fraction.

[0078] a. Before adding 250 µL of 2:1:1 water:methanol:ethanol to the Captiva EMR-Lipid plate, wash the plate with 250 µL of 2:1:1 water:methanol:ethanol to the plate that previously contained a plasma + 1:1 methanol:ethanol + water sample.

[0079] b. After initial filtration at a lower vacuum pressure, use a higher vacuum pressure to elute additional wash solution.

[0080] c. Alternatively, a positive pressure can be used to move a 2:1:1 water:methanol:ethanol solution through the plate. After an initial filtration at a lower positive pressure, a higher positive pressure can be used to elute additional wash solution.

[0081] 14. This is the end of the metabolite extraction section of the workflow.

[0082] a. It can retain the polar metabolite collection plate and dry the sample under a nitrogen flow while heating to the bottom of the plate at 30-37°C.

[0083] i. As an alternative, a vacuum concentrator can be used to dry the sample.

[0084] b. Before analysis, the dried sample can be stored at -80°C. For analysis, the sample can be redissolved in an LC / MS-compatible solvent. The sample can be shaken, mixed with a vortex mixer, or sonicated in a bath to aid dissolution. The sample can be centrifuged to bring it to the bottom of the well or remove debris. Before analysis, the sample supernatant can be transferred to a new plate or LC / MS vial.

[0085] 15. Reset Bravo to complete lipid elution. Retain the Captiva EMR-Lipid plate as it contains lipids.

[0086] 16. Add the lipid elution solution (2:1 methanol:dichloromethane) to the 96-well plate. The plate has a glass liner to prevent contaminants from leaching into the lipid elution solution.

[0087] a. As an alternative, a glass or glass-lined container can be used to hold a 2:1 methanol:dichloromethane mixture.

[0088] 17. Using Bravo, transfer 900 µL of 2:1 methanol:dichloromethane to the retained Captiva EMR-Lipid plate. Use vacuum filtration to draw the lipid elution solution through the Captiva EMR-Lipid plate. The target flow rate for lipid elution is 1 drop every 3–5 seconds.

[0089] a. Similar to the metabolite extraction section of the workflow, positive pressure can be used to propel the lipid elution solution through the Captiva EMR-Lipid plate.

[0090] 18. When there is no lipid elution solution remaining in the wells of the Captiva EMR-Lipid plate, use a higher vacuum pressure to elute any additional lipid elution solution remaining in the Captiva EMR-Lipid plate.

[0091] a. As an alternative, when there is no lipid elution solution remaining in the wells of the Captiva EMR-Lipid plate, a higher positive pressure can be used to elute any additional lipid elution solution retained in the Captiva EMR-Lipid plate.

[0092] 19. Collect the lipid eluent. This is the lipid fraction of the sample. The lipid eluent collection plate is lined with glass to prevent contaminants from leaching into the lipid-containing fraction.

[0093] 20. Perform an additional lipid elution step by passing 900 μL of 2:1 methanol:dichloromethane through a Captiva EMR-Lipid plate.

[0094] a. It is recommended to complete at least two lipid elution steps; some lipid classes show improved recovery when using up to four lipid elutions.

[0095] b. When four lipid elutions are completed, dry or partially dry the lipid elution solution after two lipid elutions to provide space in the lipid collection plate for additional lipid elution solution.

[0096] c. Optionally, post-spiking of deuterated lipids can be added to the lipid collection plate to aid in the determination of lipid recovery. Post-spiking of deuterated lipids can also be used as an internal standard.

[0097] d. Dry the retained lipid collection plate under a nitrogen stream while heating to the bottom of the plate at 30-37°C.

[0098] i. As an alternative, a vacuum concentrator can be used to dry the sample.

[0099] 21. Store the dried sample at -80°C before analysis. For analysis, redissolve the sample in an LC / MS-compatible solvent. Seal the plate, vibrate on an orbital oscillator, and gently centrifuge to bring the sample to the bottom of the wells. The sample can be mixed with a vortex mixer or sonicated in a bath to aid dissolution. The sample can be centrifuged to remove debris. Before analysis, the sample supernatant can be transferred to a new plate or LC / MS vial.

[0100] 3-in-1 (protein, metabolite, and lipid) workflow, with no protein particle washing. In short, plasma samples were mixed with a 1:1 methanol:ethanol mixture and incubated simultaneously using a bath sonication process. Proteins were separated into particles by centrifugation, the supernatant was diluted with water, and passed through a Captiva EMR-Lipid plate (Agilent Technologies). Eluents containing polar metabolites were collected. The plate was eluted with two 2:1:1 water:methanol:ethanol solutions to collect more metabolites from the elution. The plate was then eluted with a lipid elution solution (2:1 methanol:dichloromethane). Lipids were collected in the eluent. The plate was eluted again with the same lipid elution solution, and the eluents were combined with the first eluent, dried, reconstructed, and analyzed by mass spectrometry.

[0101] Automation can be added to a 3-in-1 workflow. For example: 1. Transfer 20 μL of plasma aliquots into the wells of a 96-well plate.

[0102] 2. Add approximately 130-160 μL of a 1:1 methanol:ethanol mixture to each well of a separate 96-well plate.

[0103] a. In some iterations, the 1:1 methanol:ethanol solution included a pre-spike of deuterium-labeled lipids (dissolved in 1:1 methanol:ethanol) to assess lipid recovery. The pre-spike of deuterium-labeled lipids could also be used as an internal standard.

[0104] b. As an alternative, a 1:1 methanol:ethanol ratio can be added to a single-well reservoir.

[0105] 3. Pour the water:methanol:ethanol ratio of 2:1:1 into the single-hole storage tank.

[0106] a. As an alternative, a 2:1:1 ratio of water:methanol:ethanol can be added to the wells of a 96-well plate.

[0107] 4. Add milliQ water to the 96-well plate and add 92.5 μL (112.5 μL minus the plasma sample volume) to the wells of the plate.

[0108] 5. Add the plasma plate, 1:1 methanol:ethanol plate, water plate, and 2:1:1 water:methanol:ethanol plate to the Agilent Bravo Liquid Handler equipped with supplementary protocols for processing plasma samples. All other necessary consumables (e.g., Captiva EMR-Lipid plates, metabolite collection plates, and pipette tips for liquid and sample transfer) to the Bravo as well.

[0109] 6. Using the supplementary protocol, Bravo transferred 112.5 µL of a 1:1 methanol:ethanol solution to the plate containing the plasma sample. Mix the plasma and 1:1 methanol:ethanol solution using a pipette and plate shaking. The 1:1 methanol:ethanol solution precipitates proteins from the plasma.

[0110] 7. Complete the optional 10-minute incubation to equilibrate the sample.

[0111] a. Use bath sonication during this incubation period to help equilibrate the sample.

[0112] 8. After ultrasonic treatment in a bath, the precipitated protein was separated into particles by centrifugation. The plate containing the sample was centrifuged at 2,272 × g for 60 minutes at 20°C.

[0113] 9. After the protein is granulated, transfer the sample supernatant to a plate containing 92.5 µL (112.5 µL minus the plasma sample volume) of water in each well. Mix the sample using a pipette.

[0114] 10. Complete the second optional 10-minute incubation to equilibrate the sample.

[0115] a. Bath sonication is not required in this step because protein precipitates have been removed beforehand.

[0116] 11. Transfer the sample supernatant and aqueous solution to a Captiva EMR-Lipid plate. Use vacuum pressure to draw the sample supernatant and aqueous solution through the plate.

[0117] a. After initial filtration at a lower vacuum pressure, use a higher vacuum pressure to elute additional sample solution.

[0118] b. Alternatively, positive pressure can be used to push the sample through the plate. After initial filtration at a lower positive pressure, a higher positive pressure can be used to elute additional sample solution.

[0119] 12. Collect polar metabolites from the effluent.

[0120] 13. Wash the Captiva EMR-Lipid plate with 250 μL of a 2:1:1 water:methanol:ethanol solution, and then use vacuum pressure to move the solution through the plate. Collect the eluent along with the previous eluent and add the polar metabolite fraction.

[0121] a. Before adding 250 µL of 2:1:1 water:methanol:ethanol to the Captiva EMR-Lipid plate, wash the plate, which previously contained the sample supernatant and aqueous solution, with 250 µL of 2:1:1 water:methanol:ethanol.

[0122] b. After initial filtration at a lower vacuum pressure, use a higher vacuum pressure to elute additional wash solution.

[0123] c. Alternatively, a positive pressure can be used to move a 2:1:1 water:methanol:ethanol solution through the plate. After an initial filtration at a lower positive pressure, a higher positive pressure can be used to elute additional wash solution.

[0124] 14. Wash the Captiva EMR-Lipid plate a second time with 250 μL of a 2:1:1 water:methanol:ethanol solution, and then use vacuum pressure to move the solution through the plate again. Collect the eluent together with the previous eluent and add the polar metabolite fraction.

[0125] a. Before adding 250 µL of 2:1:1 water:methanol:ethanol to the Captiva EMR-Lipid plate, wash the plate, which previously contained the sample supernatant and aqueous solution, with 250 µL of 2:1:1 water:methanol:ethanol.

[0126] b. After initial filtration at a lower vacuum pressure, use a higher vacuum pressure to elute additional wash solution.

[0127] c. Alternatively, a positive pressure can be used to move a 2:1:1 water:methanol:ethanol solution through the plate. After an initial filtration at a lower positive pressure, a higher positive pressure can be used to elute additional wash solution.

[0128] 15. This is the end of the metabolite extraction section of the workflow.

[0129] a. It can retain the polar metabolite collection plate and dry the sample under a nitrogen flow while heating to the bottom of the plate at 30-37°C.

[0130] i. As an alternative, a vacuum concentrator can be used to dry the sample.

[0131] b. Before analysis, the dried sample can be stored at -80°C. For analysis, the sample can be redissolved in an LC / MS-compatible solvent. The sample can be shaken, mixed with a vortex mixer, or sonicated in a bath to aid dissolution. The sample can be centrifuged to bring it to the bottom of the well or remove debris. Before analysis, the sample supernatant can be transferred to a new plate or LC / MS vial.

[0132] 16. Reset Bravo to complete lipid elution. Retain the Captiva EMR-Lipid plate as it contains lipids.

[0133] 17. Add the lipid elution solution (2:1 methanol:dichloromethane) to the 96-well plate. The plate has a glass liner to prevent contaminants from leaching into the lipid elution solution.

[0134] a. Alternatively, a glass or glass-lined container can be used to hold a 2:1 methanol:dichloromethane mixture.

[0135] 18. Using Bravo, transfer 900 µL of 2:1 methanol:dichloromethane to a retained Captiva EMR-Lipid plate. Use vacuum filtration to draw the lipid elution solution through the Captiva EMR-Lipid plate. The target flow rate for lipid elution is 1 drop every 3–5 seconds.

[0136] a. Similar to the metabolite extraction section of the workflow, positive pressure can be used to propel the lipid elution solution through the Captiva EMR-Lipid plate.

[0137] 19. When there is no lipid elution solution remaining in the wells of the Captiva EMR-Lipid plate, use a higher vacuum pressure to elute any additional lipid elution solution remaining in the Captiva EMR-Lipid plate.

[0138] a. As an alternative, when there is no lipid elution solution remaining in the wells of the Captiva EMR-Lipid plate, a higher positive pressure can be used to elute any additional lipid elution solution retained in the Captiva EMR-Lipid plate.

[0139] 20. Collect the lipid eluent. This is the lipid fraction of the sample. The lipid eluent collection plate is lined with glass to prevent contaminants from leaching into the lipid-containing fraction.

[0140] 21. Perform an additional lipid elution step by passing 900 μL of 2:1 methanol:dichloromethane through a Captiva EMR-Lipid plate.

[0141] a. It is recommended to complete at least two lipid elution steps; some lipid classes show improved recovery when using up to four lipid elutions.

[0142] b. When four lipid elutions are completed, dry or partially dry the lipid elution solution after the first two lipid elutions to provide space in the lipid collection plate for additional lipid elution solution.

[0143] c. Optionally, post-spiking of deuterated lipids can be added to the lipid collection plate to aid in the determination of lipid recovery. Post-spiking of deuterated lipids can also be used as an internal standard.

[0144] d. Dry the retained lipid collection plate under a nitrogen stream while heating to the bottom of the plate at 30-37°C.

[0145] i. As an alternative, a vacuum concentrator can be used to dry the sample.

[0146] 22. Store the dried sample at -80°C before analysis. For analysis, redissolve the sample in an LC / MS-compatible solvent. Seal the plate, oscillate on an orbital shaker, and gently centrifuge to bring the sample to the bottom of the wells. The sample can be mixed with a vortex mixer or sonicated in a bath to aid dissolution. The sample can be centrifuged to remove debris. Before analysis, the sample supernatant can be transferred to a new plate or LC / MS vial.

[0147] A 3-in-1 (protein, metabolite, and lipid) workflow includes protein particle washing. In short, plasma samples were mixed with a 1:1 methanol:ethanol mixture and incubated simultaneously with a sonic bath. Proteins were separated into particles by centrifugation, the supernatant was diluted with water, and passed through a Captiva EMR-Lipid plate (Agilent Technologies). The effluent containing polar metabolites was collected. The plate was eluted with two 2:1:1 water:methanol:ethanol fractions to collect more metabolites from the effluent.

[0148] Protein particles were washed with a lipid elution solution (2:1 methanol:dichloromethane), sonicated in a bath, and centrifuged again to collect the particles and wash buffer. The lipid-containing wash buffer was used to elute Captiva EMR-Lipid plates, which had additional lipids bound to them. The lipids were collected in the eluent. The plate was eluted again with the same lipid elution solution, and the eluent was combined with the first eluent, dried, reconstructed, and analyzed by mass spectrometry.

[0149] Automation can be incorporated into the new 3-in-1 workflow. For example: 1. Transfer 20 μL of plasma aliquots into the wells of a 96-well plate.

[0150] 2. Add approximately 130-160 μL of a 1:1 methanol:ethanol mixture to each well of a separate 96-well plate.

[0151] a. In some iterations, the 1:1 methanol:ethanol solution included a pre-spike of deuterium-labeled lipids (dissolved in 1:1 methanol:ethanol) to assess lipid recovery. The pre-spike of deuterium-labeled lipids could also be used as an internal standard.

[0152] b. As an alternative, a 1:1 methanol:ethanol ratio can be added to a single-well reservoir.

[0153] 3. Pour the water:methanol:ethanol ratio of 2:1:1 into the single-hole storage tank.

[0154] a. As an alternative, a 2:1:1 ratio of water:methanol:ethanol can be added to the wells of a 96-well plate.

[0155] 4. Add milliQ water to the 96-well plate and add 92.5 μL (112.5 μL minus the plasma sample volume) to the wells of the plate.

[0156] 5. Add the plasma plate, 1:1 methanol:ethanol plate, water plate, and 2:1:1 water:methanol:ethanol plate to the Agilent Bravo Liquid Handler equipped with supplementary protocols for processing plasma samples. All other necessary consumables (e.g., Captiva EMR-Lipid plates, metabolite collection plates, and pipette tips for liquid and sample transfer) to the Bravo as well.

[0157] 6. Using the supplementary protocol, Bravo transferred 112.5 µL of a 1:1 methanol:ethanol solution to the plate containing the plasma sample. Mix the plasma and 1:1 methanol:ethanol solution using a pipette and plate shaking. The 1:1 methanol:ethanol solution precipitates proteins from the plasma.

[0158] 7. Complete the optional 10-minute incubation to equilibrate the sample.

[0159] a. Use bath sonication during this incubation period to help equilibrate the sample.

[0160] 8. After ultrasonic treatment in a bath, the precipitated protein was separated into particles by centrifugation. The plate containing the sample was centrifuged at 2,272 × g for 60 minutes at 20°C.

[0161] 9. After the protein is granulated, transfer the sample supernatant to a plate containing 92.5 µL (112.5 µL minus the plasma sample volume) of water in each well. Mix the sample using a pipette.

[0162] 10. Complete the second optional 10-minute incubation to equilibrate the sample.

[0163] a. Bath sonication is not required in this step because protein precipitates have been removed beforehand.

[0164] 11. Transfer the sample supernatant and aqueous solution to a Captiva EMR-Lipid plate. Use vacuum pressure to draw the sample supernatant and aqueous solution through the plate.

[0165] a. After initial filtration at a lower vacuum pressure, use a higher vacuum pressure to elute additional sample solution.

[0166] b. Alternatively, positive pressure can be used to push the sample through the plate. After initial filtration at a lower positive pressure, a higher positive pressure can be used to elute additional sample solution.

[0167] 12. Collect polar metabolites from the effluent.

[0168] 13. Wash the Captiva EMR-Lipid plate with 250 μL of a 2:1:1 water:methanol:ethanol solution, and then use vacuum pressure to move the solution through the plate. Collect the eluent along with the previous eluent and add the polar metabolite fraction.

[0169] a. Before adding 250 µL of 2:1:1 water:methanol:ethanol to the Captiva EMR-Lipid plate, wash the plate, which previously contained the sample supernatant and aqueous solution, with 250 µL of 2:1:1 water:methanol:ethanol.

[0170] b. After initial filtration at a lower vacuum pressure, use a higher vacuum pressure to elute additional wash solution.

[0171] c. Alternatively, a positive pressure can be used to move a 2:1:1 water:methanol:ethanol solution through the plate. After an initial filtration at a lower positive pressure, a higher positive pressure can be used to elute additional wash solution.

[0172] 14. Wash the Captiva EMR-Lipid plate a second time with 250 μL of a 2:1:1 water:methanol:ethanol solution, and then use vacuum pressure to move the solution through the plate again. Collect the eluent together with the previous eluent and add the polar metabolite fraction.

[0173] a. Before adding 250 µL of 2:1:1 water:methanol:ethanol to the Captiva EMR-Lipid plate, wash the plate, which previously contained the sample supernatant and aqueous solution, with 250 µL of 2:1:1 water:methanol:ethanol.

[0174] b. After initial filtration at a lower vacuum pressure, use a higher vacuum pressure to elute additional wash solution.

[0175] c. Alternatively, a positive pressure can be used to move a 2:1:1 water:methanol:ethanol solution through the plate. After an initial filtration at a lower positive pressure, a higher positive pressure can be used to elute additional wash solution.

[0176] 15. This is the end of the metabolite extraction section of the workflow.

[0177] a. It can retain the polar metabolite collection plate and dry the sample under a nitrogen flow while heating to the bottom of the plate at 30-37°C.

[0178] i. As an alternative, a vacuum concentrator can be used to dry the sample.

[0179] b. Before analysis, the dried sample can be stored at -80°C. For analysis, the sample can be redissolved in an LC / MS-compatible solvent. The sample can be shaken, mixed with a vortex mixer, or sonicated in a bath to aid dissolution. The sample can be centrifuged to bring it to the bottom of the well or remove debris. Before analysis, the sample supernatant can be transferred to a new plate or LC / MS vial.

[0180] 16. Reset the Bravo to complete lipid elution. Retain the Captiva EMR-Lipid plate as it contains lipids. Retain the plate containing protein particles for washing the protein particles with the lipid elution solution to extract lipids from the protein particles.

[0181] 17. Add the lipid elution solution (2:1 methanol:dichloromethane) to the 96-well plate. The plate has a glass liner to prevent contaminants from leaching into the lipid elution solution.

[0182] a. Alternatively, a glass or glass-lined container can be used to hold a 2:1 methanol:dichloromethane mixture.

[0183] 18. Using Bravo, transfer 900 μL of 2:1 methanol:dichloromethane to the retained protein particles and mix the sample via pipette mixing.

[0184] a. As an alternative, sample mixing may also include plate oscillation.

[0185] 19. Sonicate the protein particle plate containing the lipid elution solution in a bath for 10 minutes.

[0186] a. During bath sonication, some protein particles were not resuspended; however, this did not appear to affect lipid recovery compared to the pores where protein particles were resuspended during bath sonication.

[0187] 20. After ultrasonic treatment in a bath, the protein precipitate was separated into particles by centrifugation. The plate was centrifuged at 2,272 × g for 60 minutes at 20°C.

[0188] 21. Using Bravo, transfer the lipid elution supernatant (i.e., wash solution) to a Captiva EMR-Lipid plate. Use vacuum filtration to draw the lipid elution solution through the Captiva EMR-Lipid plate. The target flow rate for lipid elution is 1 drop every 3–5 seconds.

[0189] a. As an alternative, positive pressure can be used to propel the lipid elution solution through the Captiva EMR-Lipid plate.

[0190] 22. When there is no lipid elution solution remaining in the wells of the Captiva EMR-Lipid plate, use a higher vacuum pressure to elute any additional lipid elution solution remaining in the Captiva EMR-Lipid plate.

[0191] a. As an alternative, when there is no lipid elution solution remaining in the wells of the Captiva EMR-Lipid plate, a higher positive pressure can be used to elute any additional lipid elution solution retained in the Captiva EMR-Lipid plate.

[0192] 23. Collect the lipid eluent. This is the lipid fraction of the sample. The lipid eluent collection plate is lined with glass to prevent contaminants from leaching into the lipid-containing fraction.

[0193] 24. Perform an additional lipid elution step by passing 900 μL of 2:1 methanol:dichloromethane through a Captiva EMR-Lipid plate.

[0194] a. It is recommended to complete at least two lipid elution steps; some lipid classes show improved recovery when using up to four lipid elutions.

[0195] b. When four lipid elutions are completed, dry or partially dry the lipid elution solution after two lipid elutions to provide space in the lipid collection plate for additional lipid elution solution.

[0196] c. Optionally, post-spiking of deuterated lipids can be added to the lipid collection plate to aid in the determination of lipid recovery. Post-spiking of deuterated lipids can also be used as an internal standard.

[0197] d. Dry the retained lipid collection plate under a nitrogen stream while heating to the bottom of the plate at 30-37°C.

[0198] i. As an alternative, a vacuum concentrator can be used to dry the sample.

[0199] 25. Store the dried sample at -80°C before analysis. For analysis, redissolve the sample in an LC / MS-compatible solvent. Seal the plate, oscillate on an orbital shaker, and gently centrifuge to bring the sample to the bottom of the wells. The sample can be mixed with a vortex mixer or sonicated in a bath to aid dissolution. The sample can be centrifuged to remove debris. Before analysis, the sample supernatant can be transferred to a new plate or LC / MS vial.

[0200] * * * According to this disclosure, all methods disclosed and claimed herein can be manufactured and performed without excessive experimentation. While the compositions and methods of the invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that changes can be made to the methods and the steps or order of steps described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant reagents can be substituted for those described herein while obtaining the same or similar results. All such similar alternatives and modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

Claims

1. A method for extracting lipids from a sample, comprising: (a) Precipitate proteins from the sample to produce a sample mixture comprising protein precipitate and supernatant, and separate the protein precipitate from the supernatant; (b) Passing the supernatant through a solid-phase extraction matrix with an affinity for lipids to produce a first effluent; (c) Wash the protein precipitate with a first lipid elution solution to obtain a washing solution; (d) Elute the solid-phase extraction matrix with a second lipid elution solution after (b) to obtain a second effluent; The washing liquid and the second effluent contain lipids.

2. The method according to claim 1 further includes: Water is added to the supernatant before it is passed through the solid-phase extraction matrix.

3. The method according to claim 1 or 2, wherein the second lipid elution solution comprises the washing solution.

4. The method according to claim 1 or 2, wherein the first lipid elution solution and the second lipid elution solution have the same composition.

5. The method according to any one of the preceding claims, further comprising: The solid-phase extraction matrix was further eluted with a lipid elution solution after (d).

6. The method according to any one of the preceding claims further comprises: Metabolites are collected from the first effluent.

7. The method according to any one of the preceding claims further comprises: Prior to (d), the solid-phase extraction matrix is ​​eluted with a metabolite elution solution to collect the metabolites in the resulting eluent.

8. The method according to claim 7, further comprising: The resulting eluent was combined with the first effluent.

9. The method according to any one of the preceding claims, further comprising: Proteins are collected from the protein precipitate.

10. The method according to any one of the preceding claims, wherein the protein precipitate is not separated from the supernatant by filtration.

11. The method according to any one of the preceding claims, wherein the protein precipitate is separated from the supernatant by centrifugation.

12. The method according to any one of the preceding claims, wherein the protein is precipitated using a solution containing methanol and ethanol.

13. The method according to any one of the preceding claims, wherein the protein is precipitated using a 50:50 methanol / ethanol (v / v) mixture.

14. The method according to any one of the preceding claims, wherein the water content of the sample mixture is 40% or less during the separation.

15. The method according to any one of the preceding claims, wherein the water content of the sample mixture is 35% or less during the separation.

16. The method according to any one of the preceding claims, wherein the water content of the sample mixture is 30% or less during the separation.

17. The method according to any one of the preceding claims, wherein the water content of the sample mixture is 25% or less during the separation.

18. The method according to any one of the preceding claims, wherein the water content of the sample mixture is 20% or less during the separation.

19. The method according to any one of the preceding claims, wherein the water content of the sample mixture is 15% or less during the separation.

20. The method according to any one of the preceding claims, wherein the water content of the sample mixture is 10% or less during the separation.

21. The method according to any one of the preceding claims, wherein the water content of the sample mixture is 5% or less during the separation.

22. The method according to any one of the preceding claims, wherein the sample is plasma.

23. The method according to any one of claims 1 to 21, wherein the sample is serum.

24. The method according to any one of claims 1 to 21, wherein the sample is a bodily fluid other than serum or plasma.

25. The method according to any one of claims 1 to 21, wherein the sample comprises lysed cells.

26. The method of claim 25, wherein the cells are derived from blood.

27. The method of claim 25, wherein the cells are cultured suspension cells.

28. The method of claim 25, wherein the cells are cultured adherent cells.

29. The method of claim 25, wherein the cell is a 3D cultured cell (e.g., an organoid, spheroid, or cell culture grown in a 3D support).

30. The method of claim 25, wherein the cells are derived from tissue.

31. The method according to any one of claims 25 to 30, wherein the sample comprises cells that have been treated with a solution containing fluorinated alcohol.

32. The method according to claim 31, wherein the fluoroalcohol comprises one or more of the following: 2,2,2-trifluoroethanol, 2,2-difluoroethanol, 2-fluoroethanol, hexafluoro-2-propanol, nonafluoro-tert-butanol, 1,1,2,2,2-pentafluoroethanol and / or 2,2,3,3,3-pentafluoro-1-propanol.

33. The method of claim 31, wherein the fluorinated alcohol is 2,2,2-trifluoroethanol.

34. The method according to any one of the preceding claims, wherein the method includes an automation step.

35. The method according to any one of the preceding claims, wherein at least one of the lipid elution solutions is a 2:1 methanol:dichloromethane.

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