A universal lipid quantification standard for use in lipidomics backgrounds

Abstract

The present disclosure provides a universal lipid quantification standard (ULQS) that includes multiple isotopically labeled lipid standards that can be used with any analytical mass spectrometry technique known in the art. The ULQS includes at least one isotopically labeled lipid species from one or more of the following lipid classes: i) phospholipids; ii) lysophospholipids; iii) cholesterol esters; iv) triacylglycerols; v) diacylglycerols; vi) ceramides; and vii) sphingomyelins.

Description

[Technical field]

[0001] The present invention relates to a universal lipid quantification standard for use in lipidomics backgrounds. [Background technology]

[0002] Lipids are naturally occurring organic molecules that can have either or both hydrophobic and hydrophilic properties (i.e. polar lipids such as phospholipids and sphingomyelins) or strictly hydrophobic properties (i.e. neutral lipids such as tri-, di-, and monoglycerides and sterol esters). Lipids are present in the cells of all organisms and play complex roles in a variety of physiological processes. Furthermore, certain lipid species are used as biomarkers for certain disease states (e.g. coronary heart disease and infectious diseases). Lipids are also present in food, and lipid monitoring can be used to verify food safety.

[0003] There is no single system or method for identifying and quantifying all lipids present in a particular sample in a reasonable amount of time. This is due, in part, to the complexity of lipids as a class of compounds. The number of actual lipid entities in living cells is enormous. Approximately 40,000 unique lipid structures are cataloged in the LIPID MAPS database. Furthermore, the dynamic range over which lipid concentrations in biological samples can vary is as large as 10 12 It could be more than that.

[0004] One aspect of lipid complexity is the presence of closely related families of lipids in a sample that differ only in the number of fatty acyl carboxyls and / or the number / position of double bonds, as well as slight variations in structure (e.g., ether / ester substitution and double bond configuration). These closely related families contain molecular species that have very similar structures and molecular weights to each other. Thus, many lipid species share the same mass (either in precursor ions and / or product ions). This phenomenon is called isobaric interference and has a significant impact on the analytical identification and quantification of individual lipid molecular species.

[0005] Mass spectrometry has emerged as a powerful tool for the analysis of all lipids. Lipidological analysis of biological systems using various approaches is now possible with quantitative measurements of thousands of lipid molecular species in a single analysis. The lipidomics field remains challenging, mainly because accurately developing quantitative analytical methods with the correct internal standard strategy is very laborious. In quantitative MS, stable isotope-labeled analogs of the analyte are used as internal standards to measure the ratio of the signal intensities of the analyte and the internal standard rather than the absolute intensities. This ratio is then converted to the analyte concentration by a calibration curve generated using a reference standard. However, this approach cannot be utilized in most lipidomics studies due to the lack of sufficient internal standards for a representative number of lipid species present in the sample. Furthermore, many internal standards for use in lipid analysis cannot correct the problems of extraction efficiency, ionization efficiency, fragmentation efficiency (due to the acyl chain length and the number of double bonds present in the acyl chain) and isobaric interference.

[0006] Thus, the absence of internal standards that reflect structural diversity (e.g., number and position of double bonds in fatty acyl chains, diversity of fatty acyl chain lengths, positional isomers) is a limiting factor in the analysis of lipids by MS. As a result, there is a clear need for internal standards for use in quantitative analysis of lipids by MS that address issues inherent to the analysis of lipids by MS, such as, but not limited to, extraction efficiency, ionization efficiency, fragmentation efficiency by acyl chain length, fragmentation efficiency by the number of double bonds present in the acyl chain, and / or isobaric interferences. Furthermore, the necessary internal standards must be equally usable with the three common analytical MS strategies, i.e., methods based on normal-phase chromatography, reversed-phase chromatography, and infusion of sample introduction into the mass spectrometer ("shotgun"). Summary of the Invention

[0007] The present disclosure provides a solution to these problems and other unmet needs in the art regarding the analysis of lipids by MS by providing a universal lipid quantification standard (ULQS) for use in lipid quantification. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows examples of design principles for selecting lipid species for inclusion in the ULQS of the present disclosure. [Figure 2A] FIG. 2A shows an example of an MS scan of PE and LPE species in a lipid sample. [Figure 2B] FIG. 2B shows an example of the relationship between endogenous PE and LPE species in the same lipid and PE and LPE species (represented by total carbon numbers in fatty acyl chains) present in one embodiment of the ULQS of the present disclosure. [Figure 2C] FIG. 2C shows an example of how endogenous lipid species in a sample are quantified using one embodiment of the ULQS of the present disclosure. [Diagram 3] FIG. 3 shows an exemplary distribution of ULQS SM and PC species determined by HILIC MS. [Figure 4] FIG. 4 shows an exemplary calibration curve of ULQS PS species determined by HILIC MS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] definition All patent applications, patents, and printed publications cited herein as being incorporated by reference are hereby incorporated by reference (unless otherwise noted), except for any definitions, subject matter disclaimers or disclaimers, and except to the extent the incorporated material contradicts the explicit disclosure of this specification, the language of the present disclosure will control.

[0010] As used herein, an "isotopic label" results in a mass shift of the labeled molecule relative to an unlabeled molecule when analyzed by mass spectrometry techniques. Examples of suitable isotopic labels include deuterium (d or2 H), 13 C, and 15 N is included, the isotopic label can be incorporated at one or more positions in the molecule, and more than one type of isotopic label can be used on the same isotopically labeled molecule. In a preferred embodiment, the isotopic labels are the same. In another preferred embodiment, the isotopic labels are the same and the isotopic label is deuterium.

[0011] As used herein, lipids are referred to according to the following nomenclature: CE is cholesteryl ester, CER is sphingomyelin, TAG is triacylglycerol, DAG is diacylglycerol, PL is phospholipid, PC is phosphatidylcholine, PE is phosphatidylethanolamine, PS is phosphatidylserine, PG is phosphatidylglycerol, PI is phosphatidylinositol, LPL is lysophospholipid, LPC is lysophosphatidylcholine, LPE is lysophosphatidylethanolamine, LPS is lysophosphatidylserine, LPG is lysophosphatidylglycerol, and LPI is lysophosphatidyllinositol.

[0012] The nomenclature X:Y indicates that X is the total number of carbon atoms in the fatty acid portion of the molecule and Y is the total number of double bonds in the fatty acid portion of the molecule. For example, CE 16:1 indicates a cholesteryl ester with an acyl chain of 16 carbon atoms containing a single double bond. Multiple X:Y designations can be used when there are more on the acyl chain. For example, TAG 16:0 / 15:1 / 16:0 represents a triacylglycerol molecule with three acyl chains, two saturated acyl chains 16 carbon atoms in length and one unsaturated acyl chain 15 carbon atoms in length with a single bond.

[0013] As used herein, the term "substantially," when referring to a particular amount or range, means at least 80% or more of such amount or range, e.g., 90% or more, 95% or more, or 98% or more.

[0014] As used herein, the term "saturated" in reference to an acyl chain means that the acyl chain does not contain any double or triple bonds.

[0015] As used herein, the term "total composition score" or "total composition" refers to the combination of the total number of carbon atoms and the total number of double bonds on all fatty acyl moieties in a lipid species (e.g., CE 16:1 can represent a cholesteryl ester with an acyl chain of 16 carbon atoms including a single double bond, or PE 39:4 can represent a PE with one acyl chain of 17 carbon atoms, no double bonds, and one acyl chain of 22 carbon atoms and 4 double bonds). The total composition score can refer to the acyl chain portion of the double bond portion individually.

[0016] As used herein, the term "unsaturated" in reference to an acyl chain means that the acyl chain contains a double or triple bond, preferably a double bond.

[0017] Terms defined herein have the meanings commonly understood by those skilled in the art in the field related to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a single entity, but include a general class of which a specific example can be used for illustration. Terms in this specification are used to describe specific embodiments of the present invention, but their use does not distinguish the present invention, except as outlined in the scope of the patent invention.

[0018] Mass Spectrometry Technology Overview The described ULQS is designed to be used with samples that are analyzed by mass spectrometry (MS). MS is carried out using a mass spectrometer, and includes an ionization source for ionizing samples (which may be fragmented before being introduced into the mass spectrometer) and generating charged molecules for further analysis. For example, the ionization of lipid species in samples can be carried out by electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), laser diode thermal desorption (LDTD), fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray / plasma spray ionization, surface-enhanced laser desorption ionization (SELDI), inductively coupled plasma (ICP) and particle beam ionization. In a preferred embodiment, samples are ionized by ESI.

[0019] A sample may be introduced directly into the mass spectrometer (e.g., in a shotgun-based method) or the components of the sample may be fractionated (e.g., in normal-phase or reverse-phase chromatography techniques) before being introduced into the mass spectrometer.

[0020] MS techniques can be performed in positive or negative ionization modes. Generally, in MS techniques, after a sample is ionized, the generated positively or negatively charged ions can be analyzed to determine the mass-to-charge ratio. Suitable analyzers for determining mass-to-charge ratio include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers. MS systems can detect ions using several detection modes. For example, selected ions can be detected using selective ion monitoring mode (SIM). In certain embodiments, mass-to-charge ratio is determined using a quadrupole analyzer. For example, in a "quadrupole" or "quadrupole ion trap" device, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between the electrodes, the amplitude of the RF signal, and the mass / charge ratio. The voltage and amplitude can be selected such that only ions with a particular mass / charge ratio travel the length of the quadrupole, while all other ions are deflected. Thus, a quadrupole instrument can act as both a "mass filter" and a "mass detector" for ions injected into the instrument.

[0021] In another embodiment, the resolution of MS techniques can be improved by using "tandem mass spectrometry", also called "MS / MS", which is used to monitor mass transitions resulting from collision-induced dissociation or neutral loss, and can be monitored, for example, using multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). In MS / MS, precursor ions (also called parent ions) generated from the lipid of interest are filtered by the MS instrument, which fragments the precursor ions to generate one or more fragment ions (also called product ions), which can then be analyzed in a second MS step. The generated ions pass through the orifice of the instrument and enter a series of three quadrupoles (Q1, Q2, and Q3). Q1 acts as a mass filter, allowing the selection of ions (i.e., selection of "precursor" ions) to pass to Q2 based on their mass-to-charge ratio (m / z). Q2 acts as a collision chamber in which precursor ions are fragmented into fragment ions. Q3 acts as a mass filter, allowing the selection of ions (i.e., fragment ions) based on their m / z. The three quadrupoles select ions with the mass-to-charge ratio of the fatty acid ion of interest. Ions with the correct mass / charge ratio are passed through the quadrupole and collided with the detector. Precursor ion selection ensures that only ions generated by a particular analyte are passed through the fragmentation chamber where fragment ions are generated by collision with atoms of an inert gas. Because both precursor and fragment ions are generated in a reproducible manner under the given ionization / fragmentation conditions, the MS / MS MRM scan mode provides reliable and most sensitive analytical results. Alternative modes of operating a tandem mass spectrometer include precursor ion and neutral loss scan modes. Such methods are known to those skilled in the art.

[0022] When an ion collides with the detector, an electronic pulse is generated and converted to a digital signal via an electron multiplier. The collected data signal is relayed to a computer, which plots the collected ion counts versus time. The area under the peak corresponding to a particular ion, or the amplitude of such a peak, can be measured and correlated to the amount of the analyte of interest. As mentioned above, the relative abundance of a given ion can be converted to an accurate quantification of the original analyte using the ULQS as an internal standard.

[0023] Lipid quantification There are many methods used to quantify lipids in a sample using MS. In general, quantification methods are classified as i) absolute quantification, ii) relative quantification, and iii) precise quantification.

[0024] Absolute quantification is based on having an internal standard and primary standard for all lipid molecules of interest (lipids to be quantified) in a sample. This approach is not possible in lipidomics due to the number of lipids present in a sample and the diversity of lipids. Relative quantification is based on using a single internal standard (e.g., for use with injection and HILIC-based analytical methods) per class of lipid analyzed. Molecular species within each lipid class are reported as area ratios relative to a single internal standard. Using this approach, the relative amounts of lipid species between two different samples can be compared (note that relative quantification does not provide the amount of lipid species).

[0025] In contrast to the above, accurate quantification is based on using multiple internal standards per lipid class to determine the amount of lipid species present in a sample.Accurate quantification at total composition / score level or molecular species level can be obtained using the ULQS described herein using various MS techniques, such as, but not limited to, hydrophilic interaction liquid chromatography, reversed phase and shotgun-based techniques.

[0026] Universal lipid quantification standard The present disclosure provides a universal ULQS that includes multiple isotope-labeled lipid internal standards. The ULQS can be used with any analytical mass spectrometry technique known in the art, including those described herein.

[0027] In a general embodiment, the ULQS comprises at least one isotopically labeled lipid species from one or more of the following lipid classes: i) the phospholipid class; ii) the lysophospholipid class; iii) the cholesterol ester class; iv) the triacylglycerol class; v) the diacylglycerol class; vi) the ceramide class; and vii) the sphingomyelin class.

[0028] Unless otherwise stated herein, reference to lipids or lipid species contained in the ULQS is understood to mean that the lipids or lipid species are isotopically labeled as described herein. In the specific embodiment described above, the ULQS comprises more than one lipid species from one or more of the seven classes described above. The ULQS can comprise two, three, four, or five or more different lipid species from one or more of the seven classes described above. The upper limit of lipid species from a given class is generally 100 or less, for example 50 or less or 25 or less.

[0029] In one embodiment, the ULQS comprises 3-50 different lipid species from one or more of the seven classes described above. For clarity, the number of lipid species from each lipid class need not be the same. For example, with respect to the seven lipid classes described above, an exemplary ULQS may comprise 15 phospholipid species, 10 lysophospholipid species, 3 cholesterol ester species, 5 triacylglycerol species, 3 diacylglycerol species, 5 ceramide species, and 5 sphingomyelin species. As another example, with reference to the seven lipid classes described above, an exemplary ULQS may comprise 20 phospholipid species, 15 lysophospholipid species, 5 triacylglycerol species, 5 diacylglycerol species, and 5 sphingomyelin species. By way of further example, with reference to the seven lipid classes above, an exemplary ULQS may include 25 phospholipid species, 15 lysophospholipid species, 3 cholesterol ester species, 9 triacylglycerol species, 5 diacylglycerol species, 5 ceramide species, and 5 sphingomyelin species.

[0030] The composition of the ULQS can be influenced by a variety of factors, including, but not limited to, the nature of the sample matrix (e.g., plasma or urine), the source of the sample matrix (e.g., human or mouse), and the identity of the lipid species being analyzed. Furthermore, when the ULQS is used in a method to determine lipid species associated with a disease, the nature of the disease may also affect the composition of the ULQS.

[0031] When selecting lipid classes and various lipid species from each class for inclusion in the ULQS, the lipid species are selected to, among other things, modify at least one of the ionization efficiency, extraction efficiency, and different fragmentation efficiency of at least one lipid species in the sample to be analyzed or tested. In some embodiments, the various lipid species from each class for inclusion in the ULQS are selected to correct different fragmentation efficiencies and at least one of the following: ionization efficiency and extraction efficiency. In some embodiments, the various lipid species from each class for inclusion in the ULQS are selected to modify both different fragmentation efficiencies and ionization efficiency and extraction efficiency. In any of the above embodiments, the various lipid species from each class for inclusion in the ULQS are further selected to cover the entire mass range or substantially the entire mass range of at least one lipid species present in the sample. In any of the above embodiments, the concentration of the various lipid species from each class of lipids selected for inclusion in the ULQS is selected to reflect the concentration of at least one lipid species present in the sample. In any of the foregoing embodiments, the various lipid species from each class for inclusion in the ULQS are further selected to cover the entire mass range of at least one lipid species present in the sample, and the concentration of the at least one lipid species is selected to reflect the concentration of the lipid species present in the sample.

[0032] In general, the concentration of lipid species of each class contained in the ULQS is 1 to 1,000 μg / ml, preferably 1 to 500 μg / ml. The concentrations of lipid species between lipid classes may be the same or different. The concentrations of lipid species within a lipid class may be the same or different. In one embodiment, the concentration of lipid species from the phospholipid and triacylglycerol classes contained in the ULQS is 1 to 1,000 μg / ml, preferably 1 to 500 μg / ml, more preferably 10 to 250 μg / ml, even more preferably 15 to 200 μg / ml. In one embodiment, the concentration of lipid species from the lysophospholipid class contained in the ULQS is 1 to 500 μg / ml, preferably 10 to 200 μg / ml, more preferably 15 to 150 μg / ml, even more preferably 20 to 100 μg / ml. In one embodiment, the ULQS contains lipid species from the ceramide, diacylglycerol, sphingomyelin, and cholesterol ester classes at concentrations between 1 and 500 μg / ml, preferably between 10 and 350 μg / ml, more preferably between 15 and 250 μg / ml, and even more preferably between 20 and 150 μg / ml. In one embodiment, the concentrations of lipid species from the phospholipid, triacylglycerol, lysophospholipid, ceramide, diacylglycerol, sphingomyelin, and cholesterol ester classes are as set out in Table 8 herein.

[0033] In certain embodiments, ionization efficiency, extraction efficiency, and differential fragmentation efficiency are influenced by the nature of the sample matrix, the source of the sample matrix, and the identity of the lipid species being analyzed.

[0034] Suitable mass ranges (total composition scores) of a typical human sample for the various lipid classes listed above are: i) phospholipids-27:1 to 43:4 (preferably 31:1 to 39:4), ii) lysophospholipids-11:0 to 23:0 (preferably 15:0 to 19:0), iii) cholesterol esters-11:1 to 25:4 (preferably 14:1 to 22:4), iv) triacylglycerols-43:1 to 55:2 (preferably 47:1 to 51:2), v) diacylglycerols-27:1 to 43:4 (preferably 31:1 to 39:4), vi) ceramides-12:1 to 28:1 (preferably 16:1 to 24:1), and vii) sphingomyelins-12:1 to 28:1 (preferably 16:1 to 24:1).

[0035] The ULQS internal strategy design approach can be visualized as shown in Figure 1. Each lipid species in the ULQS for a particular lipid class defines a quantification window (or bucket). Each lipid species in a sample is assigned to a specific quantification window for quantification. In the case of no exact match, a best approximation is used. In FIG. 1, the ULQS includes five lipid species of the PC class designated as PC(D517:0 / 14:1), PC(D517:0 / 16:1), PC(D517:0 / 18:1), PC(D517:0 / 20:3), and PC(D517:0 / 22:4). The five PC species define a quantification window to which the lipid species in the sample are assigned. Three lipid species in the sample are shown as PC(16:0 / 18:1), PC(16:0 / 18:3), and PC(18:0 / 22:4) for illustrative purposes. None of the lipid species in the sample exactly fit the defined quantification window defined by the ULQS lipid species, so a best approximation is used. In this example, PC(16:0 / 18:1) lipids in the sample are assigned to a quantification window defined by the PC(D517:0 / 16:1) QULS species, PC(16:0 / 18:3) lipids in the sample are assigned to a quantification window defined by the PC(D517:0 / 20:3) QULS species, and PC(16:0 / 22:4) lipids in the sample are assigned to a quantification window defined by the PC(D517:0 / 22:4) QULS species.

[0036] An exemplary design strategy for the design of ULQS is shown in Figures 2A-2C, which show the design strategy for LPEs and PEs. The same approach can be used to design components of ULQS for other lipid species. In Figure 2A, an MS scan (intensity vs. precursor ion Da) of an exemplary lipid sample is shown, showing the various LPE and PE species present in the sample. A schematic over the various species shows the location and relative abundance of each LPE and PE species in a particular sample. Figure 2B shows the same MS scan shown in Figure 2A, modified to show the presence of the exemplary ULQS LPE and PE species (i.e., internal standards) for the LPE and PE species in terms of the total composition score. In this example, the ULQS LPE species are LPE 15:0, LPE 17:0, and LPE 19:0 with total composition scores of 15:0, 17:0, and 19:0, respectively, and the ULQS PE species are PE(D517:0 / 14:1), PE(D517:0 / 16:1), PE(D517:0 / 18:1), PE(D517:0 / 20:3), and PE(D517:0 / 22:4) with total composition scores of 31:1, 33:1, 35:1, 37:3, and 39:4, respectively. Figure 2B also shows the structure of the ULQS PE 31:1 standard showing the MRM transitions of the 17:0 fatty acyl moiety (787.6 / 269.3) and the 14:1 fatty acyl moiety (787.6 / 323.3). The MRM transitions of any lipid species present in the ULQS can be determined by those skilled in the art. Based on the molecular weight of the deuterated species, minimal isobaric interference with endogenous lipid species is expected. In this example, the PE class has five species and the LPE class has three species (the number of species for each class can be increased or decreased, as discussed herein), with the selection of PE and LPE species selected to account for the ionization efficiency, extraction efficiency, and differential fragmentation efficiency of the PE and LPE lipid species in the sample and to cover substantially the entire mass range of the PE and LPE lipid species in the sample. Furthermore, the incorporation of odd-chain fatty acids in the PE and LPE internal standards minimizes isobaric overlap and improves MRM specificity. Finally, the concentration of each internal standard present in the ULQS is selected to reflect the relative abundance of endogenous lipid species within each class present in the sample.

[0037] FIG. 2C shows how ULQS species are used to quantify the various PE lipid species present in a sample. As described above, each ULQS PE species defines a quantification window, and the various PE lipid species in the sample are assigned to the quantification window based on exact match or best approximation. In this example, the quantification window for each ULQS PE species is shown in alternating non-sharded and sharded boxes, and the various PE lipid species in the sample that fall within the various boxes are quantified using the specified ULQS PE quantification window. The same approach can be used to quantify LPE lipid species (as well as any other lipid species present in a sample in the presence of the appropriate ULQS internal standard).

[0038] By distributing the internal standards across the mass range of each class, it is possible to standardize the ionization efficiency, extraction efficiency, and different fragmentation efficiency of lipid species in a sample based on fatty acid / molecular weight.Furthermore, since the hydrophobicity of lipid species (both ULQS species and lipid species present in a sample) is approximately proportional to molecular weight / fatty acid chain length, the ULQS internal standards selected as described herein are distributed along the gradient (e.g., in reversed-phase analysis) in the same manner as the lipid species in the sample, thereby improving quantification accuracy.

[0039] The resulting ULQS can be used for accurate quantification of lipid species in a sample. Accurate quantification is important for a variety of reasons. First, accurate quantification of lipids in a sample provides standardization of results, allowing for comparison of data generated between different laboratories, and allows for comparison of concentrations of different lipid species in samples. Such standardization improves the ability of researchers to collaborate and share data. Additionally, accurate quantification has clinical applications, allowing for the establishment of reference values ​​for various lipid species, and allows for the use of lipid species as biomarkers in diagnostic applications.

[0040] In one embodiment, lipid species for inclusion in a ULQS include between 3 and 50 different lipid species from one or more of the seven classes described above. In certain embodiments, an exemplary ULQS may include: i) 15 phospholipid species, 10 lysophospholipid species, 3 cholesterol ester species, 5 triacylglycerol species, 3 diacylglycerol species, 3 ceramide species, and 3 sphingomyelin species; ii) 20 phospholipid species, 15 lysophospholipid species, 5 cholesterol ester species, 9 triacylglycerol species, 5 diacylglycerol species, 5 ceramide species, and 5 sphingomyelin species; or iii) 25 phospholipid species, 15 lysophospholipid species, 5 cholesterol ester species, 9 triacylglycerol species, 5 diacylglycerol species, 5 ceramide species, and 5 sphingomyelin species. Regardless of the composition of the ULQS, the individual lipid species selected for inclusion in the ULQS are selected to, among other things, modify at least one of the ionization efficiency, extraction efficiency, and differential fragmentation efficiency of at least one lipid species in the sample being analyzed / tested. In some embodiments, the individual lipid species selected for inclusion are selected to modify at least one of the differential fragmentation efficiency, ionization efficiency, and extraction efficiency (preferably both ionization efficiency and extraction efficiency). In some embodiments, the individual lipid species selected for inclusion in the ULQS are selected to modify at least one of the ionization efficiency, extraction efficiency, and differential fragmentation efficiency of at least one lipid species in the sample being tested, and the various lipid species for inclusion in the ULQS from each class are further selected to i) cover the entire mass range or substantially the entire mass range of at least one lipid species present in the sample, ii) mirror the concentration of at least one lipid species present in the sample, or iii) cover the entire mass range or substantially the entire mass range of at least one lipid species present in the sample and mirror the concentration of at least one lipid species present in the sample.

[0041] In certain embodiments, the individual lipid species selected for inclusion in the ULQS are selected to modify the fractionation fragmentation efficiency of at least one lipid species in the sample being tested, and at least one of the ionization efficiency and extraction efficiency (preferably both ionization efficiency and extraction efficiency) of at least one lipid species in the sample being analyzed / tested, and the various lipid species for inclusion in the ULQS from each class are further selected to: i) cover the entire mass range or substantially the entire mass range of at least one lipid species present in the sample; ii) mirror the concentration of at least one lipid species present in the sample; or iii) cover the entire mass range or substantially the entire mass range of at least one lipid species present in the sample and mirror the concentration of at least one lipid species present in the sample.

[0042] Phospholipids In one embodiment, the phospholipid class comprises a plurality of phospholipid species, each phospholipid species comprising an isotopic label. Any combination of phospholipid species described herein can be used in the ULQS. In one embodiment, the phospholipid class comprises one or more phospholipid species selected from the group consisting of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), and phosphatidylinositol (PI). In each of the above, the acyl chain of the phospholipid species is independently selected from a saturated C3-C30 chain, or an unsaturated C3-C30 acyl chain comprising 1-6 double bonds, such as 1-2 or 1-4 double bonds.

[0043] In one embodiment, the acyl chains of the phospholipid species are each independently a saturated or unsaturated C3-C6 acyl chain, a saturated or unsaturated C7-C11 acyl chain, a saturated or unsaturated C12-C21 acyl chain, or a saturated or unsaturated C22-C30 acyl chain. Such acyl chains may be saturated or unsaturated, regardless of length, including both even and odd chain lengths. In certain embodiments, the acyl chain lengths of the phospholipid species are each selected such that the total composition score of the acyl chain is an odd number. In certain embodiments, the acyl chains of the phospholipid species are each a 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon acyl chain, each of which is saturated or unsaturated. In another particular embodiment, the acyl chain of the phospholipid species is a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon acyl chain, each of which is saturated or unsaturated. In yet another particular embodiment, the acyl chain of the phospholipid species is a 15, 16, 17, 18, 19, 20, 21, 22, or 23 carbon acyl chain, each of which is saturated or unsaturated.

[0044] In one embodiment, one or both of the acyl chains of the phospholipid species are saturated. In another embodiment, one or both of the acyl chains of the phospholipid species are unsaturated. In yet another embodiment, one of the acyl chains is saturated and one of the acyl chains is unsaturated. When one or both of the acyl chains of the phospholipid species are unsaturated, the acyl chains can contain 1-6, 1-4, or 1-3 double bonds. In one embodiment, at least one of the acyl chains is unsaturated and contains 1-4, 1-3, or 1-2 double bonds. In some embodiments, at least one of the acyl chains contains two or more double bonds (e.g., contains 2-4 double bonds) when the acyl chain length is 18 or more or 20 or more. The double bonds in such unsaturated acyl chains may be in the cis or trans configuration, or may be a mixture of cis and trans configurations when more than one double bond is present.

[0045] In one embodiment, when multiple species of a particular phospholipid (e.g., multiple phosphatidylcholine species) are present, the acyl chains on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-4 double bonds). In one embodiment, when multiple species of phospholipid are present, the acyl chains on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-4 double bonds), with at least one acyl chain having 2-4 double bonds.

[0046] In some embodiments, when multiple species of a particular phospholipid (e.g., multiple phosphatidylcholine species) are present, the acyl chain lengths of the different species are different from each other. In some embodiments, when multiple species of a particular phospholipid (e.g., multiple phosphatidylcholine species) are present, the acyl chain lengths of the different species are different from each other, with one of the acyl chains being unsaturated (e.g., containing 1-4 double bonds) and the other acyl chain being saturated (containing no double bonds).

[0047] In any of the foregoing embodiments, each phospholipid species contains at least one isotopically labeled H atom, preferably 2 to 5 isotopically labeled H atoms. In a preferred embodiment, the isotopically labeled H atom is deuterium (D).

[0048] In one such embodiment, the phospholipid species for inclusion in the ULQS has the general formula I:

[0049] [ka]

[0050] or a pharma- ceutically acceptable salt thereof, wherein R1 is a head group moiety; R2 is a C3-C30 saturated or unsaturated acyl chain; R3 is a C3-C30 saturated or unsaturated acyl chain; and R4 is independently H or an isotope of H, provided that at least one of R4 is an isotope of H.

[0051] R1 may be selected from any phospholipid head group known in the art. Suitable choices for R1 include -(CH2) n -N(CH3)3, -(CH2) n -NH3, -(CH2) n -C(H)(NH3)-C(O)-O-, -(CH2) n These include, but are not limited to, -CH(OH)-CH(OH), inositol, and H (wherein n is independently selected from 1 to 6, preferably 1 to 2).

[0052] Particularly preferred substituents for R are of the following formulae (A), (B), (C), (D) and (E):

[0053] [ka]

[0054] When R1 is (A), the phospholipid is phosphatidylcholine (PC). When R1 is (B), the phospholipid is phosphatidylethanolamine (PE). When R1 is (C), the phospholipid is phosphatidylserine (PS). When R1 is (D), the phospholipid is phosphatidylglycerol (PG). When R1 is (E), the phospholipid is phosphatidylinositol (PI).

[0055] The following descriptions of R2, R3, and R4 are applicable to phospholipid species containing the head groups described herein.

[0056] R2 is a saturated or unsaturated acyl chain between 3 and 30 carbons in length. In one embodiment, R2 is an acyl chain between 3 and 6 carbons in length, an acyl chain between 7 and 11 carbons in length, an acyl chain between 12 and 21 carbons in length, or an acyl chain between 22 and 30 carbons in length. In a particular embodiment, R2 is an acyl chain between 10 and 25 carbons in length. In another particular embodiment, R2 is an acyl chain between 14 and 22 carbons in length. Such acyl chains, regardless of length, include both even and odd chain lengths and can be saturated or unsaturated. In a particular embodiment, R2 is an acyl chain having an even number of carbon atoms. In another embodiment, R2 is a 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon acyl chain that is saturated or unsaturated. In another embodiment, R2 is a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon acyl chain that is saturated or unsaturated. In another embodiment, R2 is a 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon acyl chain that is saturated or unsaturated.

[0057] In one embodiment, R2 is a saturated acyl chain. In another embodiment, R2 is an unsaturated acyl chain. When R2 is an unsaturated acyl chain, the acyl chain can contain 1-6, 1-4, or 1-3 double bonds. In one embodiment, R2 is an unsaturated acyl chain containing 1-4, 1-3, or 1-2 double bonds. In some embodiments, R2 contains two or more double bonds (e.g., 2-4 double bonds) when the acyl chain length is 18 or more or 20 or more. The double bonds in such unsaturated acyl chains may be in cis or trans configuration, or may be a mixture of cis and trans configurations when two or more double bonds are present.

[0058] In one embodiment, when multiple species of a particular phospholipid (e.g., multiple phosphatidylcholine species) are present, the R2 groups on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-4 double bonds). In one embodiment, when multiple species of phospholipid are present, the R2 groups on the various species are selected to include only unsaturated acyl chains (e.g., having 1-4 double bonds). In one embodiment, when multiple species of phospholipid are present, the R2 groups on the various species are selected to include only unsaturated acyl chains (e.g., having 1-4 double bonds), including acyl chains with 2-4 double bonds when the acyl chain length is 18 or more or 20 or more.

[0059] In some embodiments, the length of the acyl chain of R2 is different from the length of the acyl chain of R3, in which R2 is unsaturated (e.g., contains 1-4 double bonds, and particularly contains 2-4 double bonds when the acyl chain length is 18 or more or 20 or more), and R3 is saturated (does not contain any double bonds).

[0060] R3 is a saturated or unsaturated acyl chain between 3 and 30 carbons in length. In one embodiment, R3 is an acyl chain between 3 and 5 carbons in length, an acyl chain between 6 and 10 carbons in length, an acyl chain between 11 and 20 carbons in length, or an acyl chain between 21 and 30 carbons in length. In a particular embodiment, R3 is an acyl chain between 10 and 20 carbons in length. In another particular embodiment, R3 is an acyl chain between 15 and 20 carbons in length. Such acyl chains, regardless of length, include both even and odd chain lengths and can be saturated or unsaturated. In a particular embodiment, R3 is an acyl chain having an odd number of carbon atoms. In one embodiment, the acyl chain is saturated. In another embodiment, R3 is a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon acyl chain that is saturated or unsaturated. In another embodiment, R3 is a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon acyl chain that is saturated or unsaturated. In another embodiment, R3 is a 15, 16, 17, 18, 19, or 20 carbon acyl chain that is saturated or unsaturated.

[0061] In one embodiment, R3 is a saturated acyl chain. In another embodiment, R3 is an unsaturated acyl chain. When R3 is an unsaturated acyl chain (regardless of length), the acyl chain can contain 1-4 or 1-3 double bonds. In one embodiment, R3 is an unsaturated acyl chain containing 1 or 2 double bonds. The double bonds in such unsaturated acyl chains can be in the cis or trans configuration, or can be a mixture of cis and trans configurations when more than one double bond is present.

[0062] In one embodiment, when multiple species of phospholipid are present, the R3 groups on the various species are selected to contain only saturated acyl chains.

[0063] In some embodiments, the length of the acyl chain of R3 is different from the length of the acyl chain of R2. In some embodiments, the length of the acyl chain of R3 is different from the length of the acyl chain of R2, and R3 is saturated (no double bonds) and R2 is unsaturated (e.g., containing 1-4 double bonds, particularly 2-4 double bonds when the acyl chain length is 18 or more or 20 or more).

[0064] In one embodiment of any of the foregoing, the isotopically labeled H is deuterium (D). In one embodiment of any of the foregoing, at least one, at least two, at least three, at least four, or all five of R4 are isotopically labeled H, preferably deuterium (D).

[0065] In some embodiments, at least a portion of the phospholipid species in the ULQS have the same R3 group but different R2 groups. In some embodiments, at least a portion of the phospholipid species in the ULQS have the same R3 group but different R2 groups, where at least one or all of the R2 groups are unsaturated (e.g., containing 1-4 double bonds, especially 2-4 double bonds when the acyl chain length is 18 or greater or greater than 20).

[0066] In some embodiments, the phospholipid species in the ULQS have head groups of formulae (A), (B), (C), (D), and (E). In some embodiments, the phospholipid species in the ULQS have head groups of formulae (A), (B), (C), (D), and (E), and the phospholipid species have the same R3 group but different R2 groups. In some embodiments, the phospholipid species in the ULQS have head groups of formulae (A), (B), (C), (D), and (E), and the phospholipid species have the same R3 group but different R2 groups, where at least one or all of the R2 groups are unsaturated (e.g., containing 1-4 double bonds, especially 2-4 double bonds when the acyl chain length is 18 or greater or greater than 20).

[0067] In one embodiment, the phospholipid species included in the ULQS are at least one of each of PC, PE, PS, PG, and PI, and the phospholipid species have the same R3 group but different R2 groups, where the R3 group is of length n and the R2 group is of length n-5 to n+7, and where at least one or all of the R2 groups are unsaturated (e.g., containing 1-4 double bonds, particularly 2-4 double bonds when the acyl chain length is greater than 18 or greater than 20). In one aspect of this embodiment, n is 12-21.

[0068] In one embodiment, the phospholipid species included in the ULQS are at least one of each of PC, PE, PS, PG, and PI, where at least one of R4 (preferably all of R4) is deuterium (D), R3 is a saturated acyl chain of length n carbons, and R2 is an unsaturated acyl chain of length n-5 to n+7 carbons, where n is 12 to 21, where R2 contains 1 or 2 double bonds (preferably 1 double bond) if R2 is n-5 to n+1 in length, R2 contains 2 or 3 double bonds (preferably 3 double bonds) if R2 is n+2 to n+4 in length, and R2 contains 3 or 4 double bonds (preferably 4 double bonds) if R2 is n+5 to n+7 in length. In one aspect of this embodiment, 3 to 5 species are present for each of PC, PE, PS, PG, and PI, and each of the 15 to 25 species is different. In another aspect of this embodiment, 3-5 species are present for each PC, PE, PS, PG, and PI species, each of 15-25 species is different, and the R3 and R2 groups are repeated for each PC, PE, PS, PG, and PI species. For example, if the five PC species have a 16 carbon saturated acyl chain for R3 and 13, 15, 17, 19, and 21 carbon unsaturated acyl chains for R2, the five PE, PS, PG, and PI species also have a 16 carbon saturated acyl chain for R3 and 13, 15, 17, 19, and 21 carbon unsaturated (with the same number of double and / or triple bonds in the same positions) acyl chains for R2.

[0069] In one embodiment, the phospholipid species included in the ULQS are PC, PE, PS, PG and PI, where at least one of R4 (preferably all of R4) is deuterium (D), R3 is a saturated acyl chain of length n carbons, and R2 is an unsaturated acyl chain of length n-5 to n+7 carbons, where n is 13, 15, 17, 19, or 21, where R2 contains 1 or 2 double bonds (preferably 1 double bond) if R2 is n-5 to n+1 in length, R2 contains 2 or 3 double bonds (preferably 3 double bonds) if R2 is n+2 to n+4 in length, and R2 contains 3 or 4 double bonds (preferably 4 double bonds) if R2 is n+5 to n+7 in length. In one aspect of this embodiment, 3 to 5 species are present for each PC, PE, PS, PG, and PI species, and each of the 15 to 25 species is different. In another aspect of this embodiment, 3-5 species are present for each PC, PE, PS, PG, and PI species, each of 15-25 species is different, and the R3 and R2 groups are repeated for each PC, PE, PS, PG, and PI species. For example, if five PC species have a saturated acyl chain of 15 carbons in length for R3, and unsaturated acyl chains of 12, 14, 16, 18, and 20 carbons in length for R2, then the five PE, PS, PG, and PI species also have a saturated acyl chain of 15 carbons for R3, and acyl chains of 12, 14, 16, 18, and 20 carbons for R2 (with the same number of double and / or triple bonds in the same positions).

[0070] In one embodiment, the phospholipid species included in the ULQS are PC, PE, PS, PG and PI, where at least one of R4 (preferably all of R4) is deuterium (D), R3 is a saturated acyl chain of length n carbons, and R2 is an unsaturated acyl chain of length n-5 to n+7 carbons, where n is 15, 17, or 19, where R2 contains 1 or 2 double bonds (preferably 1 double bond) if R2 is n-5 to n+1 in length, R2 contains 2 or 3 double bonds (preferably 3 double bonds) if R2 is n+2 to n+4 in length, and R2 contains 3 or 4 double bonds (preferably 4 double bonds) if R2 is n+5 to n+7 in length. In one aspect of this embodiment, 3 to 5 species are present for each PC, PE, PS, PG, and PI species, and each of the 15 to 25 species is different. In another aspect of this embodiment, 3-5 species are present for each PC, PE, PS, PG, and PI species, each of 15-25 species being different, and the R3 and R2 groups being repeated for each PC, PE, PS, PG, and PI species. For example, if five PC species have a saturated acyl chain of 17 carbons in length for R3, and unsaturated acyl chains of 14, 16, 18, 20, and 22 carbons in length for R2, then the five PE, PS, PG, and PI species also have a saturated acyl chain of 17 carbons in length for R3, and acyl chains of 14, 16, 18, 20, and 22 carbons in length for R2 (with the same number of double and / or triple bonds in the same positions).

[0071] In one embodiment, the phospholipid species included in the ULQS can be represented by formulas IB-IF below.

[0072] [ka]

[0073] wherein for each of the compounds of formulas IB-IF, R3 is a saturated acyl chain of length n, where n is 15, 17, or 19, and for each length of n, at least one (preferably all) of R4 is deuterium, and R2 is represented by i) lengths of n-5, n-3, n+1, n+1, and n+3 carbons; ii) lengths of n-3, n-1, n+1, n+3, and n+5 carbons; or iii) five different unsaturated acyl chains of lengths of n-1, n+1, n+3, n+5, and n+7 carbons; The n-1, n+1, and n+3 carbon acyl chains contain one double bond, the n+3 acyl chain contains three double bonds, and the n+5 and n+7 acyl chains contain four double bonds.

[0074] In specific embodiments, phospholipid species of formulas IB-IF included in the ULQS include those listed in Table 1 below, where at least one of R4 (preferably all of R4) is deuterium, PC is a compound of formula IB, PE is a compound of formula IC, PS is a compound of formula ID, PG is a compound of formula IE, PI is a compound of formula IF, and the underlined portions correspond to the R2 variables and the double underlined portions correspond to the R3 variables.

[0075] [Table 1]

[0076] Lysophospholipids In one embodiment, the lysophospholipid class comprises a plurality of lysophospholipid species, wherein each lysophospholipid species comprises an isotopic label. Any combination of lysophospholipid species described herein can be used in the ULQS. In one embodiment, the lysophospholipid class comprises one or more of the following lysophospholipid species: lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylphosphatidylphosphoserine (LPS), lysophosphatidylglycerol (LPG), and lysophosphatidylinositol (LPI). In each of the above, the acyl chain of the lysophospholipid is independently selected from a saturated C3-C25 acyl chain, or an unsaturated C3-C25 acyl chain containing 1-6 double bonds.

[0077] In each of the above, the acyl chain of the lysophospholipid species is independently selected from saturated or unsaturated C3-C25 acyl chains containing 1-4 double bonds, such as 1-3 or 1-2 double bonds.

[0078] In one embodiment, the acyl chains of the lysophospholipid species are each independently a saturated or unsaturated C3-C6 acyl chain, a saturated or unsaturated C7-C11 acyl chain, a saturated or unsaturated C12-C18 acyl chain, or a saturated or unsaturated C19-C25 acyl chain. Such acyl chains can be saturated or unsaturated, regardless of length, including both even and odd chain lengths. In certain embodiments, the acyl chains of the lysophospholipid species are each selected such that the total composition score of the acyl chain is an odd number. In certain embodiments, the acyl chains of the lysophospholipid species are each a 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon acyl chain, each of which is saturated or unsaturated. In another particular embodiment, the acyl chains of the lysophospholipid species are each independently a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon acyl chain that is saturated or unsaturated. In yet another particular embodiment, the acyl chains of the lysophospholipid species are each independently a 13, 14, 15, 16, 17, 18, 19, 20, or 21 carbon acyl chain that is saturated or unsaturated.

[0079] In one embodiment, the acyl chain of the lysophospholipid species is saturated. In another embodiment, the acyl chain of the phospholipid species is unsaturated. When the acyl chain of the lysophospholipid species is unsaturated, the acyl chain can contain 1-6, 1-4, or 1-3 double bonds. In one embodiment, the acyl chain is unsaturated and contains 1-4, 1-3, or 1-2 double bonds. In certain embodiments, when the acyl chain length is 18 or more or 20 or more (e.g., contains 2-4 double bonds), the acyl chain contains two or more double bonds. The double bonds in such unsaturated acyl chains may be in the cis or trans configuration, or may be a mixture of cis and trans configurations when two or more double bonds are present.

[0080] In one embodiment, when multiple species of a particular lysophospholipid (e.g., multiple lysophosphatidylcholine species) are present, the acyl chains on the various species are selected to include only saturated acyl chains. In one embodiment, when multiple species of lysophospholipid are present, the acyl chains on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-4 double bonds).

[0081] In certain embodiments, when multiple species of a particular lysophospholipid are present (eg, multiple lysophosphatidylcholine species), the acyl chain lengths of the different species differ from one another.

[0082] In any of the foregoing embodiments, each lysophospholipid species comprises at least one isotopically labeled H atom, preferably 2 to 5 isotopically labeled H atoms. In a preferred embodiment, the isotopically labeled H atom is deuterium (D).

[0083] In one such embodiment, the phospholipid for inclusion in the ULQS has the general formula II:

[0084] [ka]

[0085] wherein: R1 is a head group moiety; R2 is a C2-C24 saturated or unsaturated acyl chain; R4 is independently H or an isotope of H, provided that at least one of R4 is an isotope of H.

[0086] R1 can be selected from any lysophospholipid head group known in the art. Suitable choices for R1 include, but are not limited to, -(CH2) n -N(CH3)3, -(CH2) n -NH3, -(CH2) n -C(H)(NH3)-C(O)-O-, -(CH2) n-CH(OH)-CH(OH), inositol, and H are included, and n is independently selected from 1 to 6, preferably 1 to 2.

[0087] Particularly preferred substituents for R1 are those of formula (A), (B), (C), (D) and (E) as defined above for phospholipids. When R1 is (A), the lysophospholipid is lysophosphatidylcholine (LPC). When R1 is (B), the lysophospholipid is lysophosphatidylethanolamine (LPE). When R1 is (C), the lysophospholipid is lysophosphatidylserine (LPS). When R1 is (D), the lysophospholipid is lysophosphatidylglycerol (LPG). When R1 is (E), the lysophospholipid is lysophosphatidylinositol (LPI).

[0088] The following description of R2 and R4 is applicable to phospholipid species containing any head group described herein.

[0089] R2 is a saturated or unsaturated acyl chain having 3 to 25 carbons. In one embodiment, R2 is an acyl chain having a length of 3 to 6 carbons, 7 to 11 carbons, 12 to 18 carbons, or 19 to 25 carbons. In a particular aspect, R2 is an acyl chain having a length of 10 to 20 carbons. Such acyl chains, regardless of length, include both even and odd chain lengths and can be saturated or unsaturated. In a particular embodiment, R2 is an acyl chain having an even number of carbon atoms. In one embodiment, R2 is an acyl chain of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbons that is saturated or unsaturated. In one embodiment, R2 is an acyl chain of 2, 3, 4, 5, 6, 7, or 8 carbons that is saturated or unsaturated. In another embodiment, R2 is a 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 carbon acyl chain, saturated or unsaturated. In another embodiment, R2 is a 14, 15, 16, 17, 18, 19, or 20 carbon acyl chain, saturated or unsaturated. In a preferred embodiment, R2 is a saturated acyl chain, where the acyl chain of R2 is of the length specified above.

[0090] In one embodiment, R2 is a saturated acyl chain. In another embodiment, R2 is an unsaturated acyl chain. In a preferred embodiment, R2 is a saturated acyl chain. When R2 is an unsaturated acyl chain, the acyl chain can contain 1-6, 1-4, or 1-3 double bonds. In one embodiment, R2 is an unsaturated acyl chain containing 1-4, 1-3, or 1-2 double bonds. The double bonds in such unsaturated acyl chains may be in the cis or trans configuration, or may be a mixture of cis and trans configurations when more than one double bond is present.

[0091] In one embodiment, when multiple species of lysophospholipid are present, the R2 groups on the various species are selected to include only saturated acyl chains and acyl chains with an odd number of carbon atoms.

[0092] In one embodiment of any of the foregoing, the isotopically labeled H is deuterium (D). In one embodiment of any of the foregoing, at least one, at least two, at least three, at least four, or all five of R4 are isotopically labeled H, preferably deuterium (D).

[0093] In some embodiments, at least some of the lysophospholipid species in the ULQS have different R2 groups. In some embodiments, the lysophospholipid species in the ULQS have head groups of formulae (A), (B), (C), (D) and (E). In some embodiments, the lysophospholipid species in the ULQS have head groups of formulae (A), (B), (C), (D) and (E), and the R2 group of the lysophospholipid species is saturated. In some embodiments, the lysophospholipid species in the ULQS have head groups of formulae (A), (B), (C), (D) and (E), and the R2 group of the lysophospholipid species is saturated, and the R2 group comprises an acyl chain having an odd number of carbon atoms.

[0094] In some embodiments, the lysophospholipid species included in the ULQS have each of the head groups of formula (A), (B), (C), (D) and (E), and the R2 group is repeated for lysophospholipid species with different head groups and for lysophospholipid species with the same head group. For example, a set of 20 lysophospholipid species suitable for inclusion in the ULQS can be represented by formula II, where there are four lysophospholipid species for each of the head groups A to E above, at least one of R4 (preferably all R4) is deuterium, and R2 for each of the four lysophospholipid species for each of the head groups A to E is a saturated acyl chain of 13, 15, 17 and 19 carbons in length.

[0095] In one embodiment, the lysophospholipid species included in the ULQS include LPC, LPE, LPS, LPG, and LPI, R2 is a saturated acyl chain of 10-25 carbons in length, and at least one of R4 (preferably all R4) is deuterium (D). In one aspect of this embodiment, 3-5 species are present for each of LPC, LPE, LPS, LPG, and LPI, and each of the 15-25 individual species is different. In another aspect of this embodiment, 3-5 species are present for each of LPC, LPE, LPS, LPG, and LPI, and each of the 15-25 individual species is different, and the R2 group is repeated for each of LPC, LPE, LPS, LPG, and LPI. For example, if the three LPC species have saturated acyl chains of 12, 14, and 16 carbons, then the three LPE, LPS, LPG, and LPI species also have saturated acyl chains of 12, 14, and 16 carbons.

[0096] In one embodiment, the lysophospholipid species included in the ULQS include LPC, LPE, LPS, LPG, and LPI, R2 is a saturated acyl chain of 11, 13, 15, 17, 19, 21, or 23 carbons in length, and at least one of R4 (preferably all R4) is deuterium (D). In one aspect of this embodiment, 3-5 species are present for each of LPC, LPE, LPS, LPG, and LPI, and each of the 15-25 species are different. In another aspect of this embodiment, 3-5 species are present for each of LPC, LPE, LPS, LPG, and LPI, and each of the 15-25 individual species are different, and the R2 group is repeated for each of LPC, LPE, LPS, LPG, and LPI. For example, if the three LPC species have saturated acyl chains of 15, 17 and 19 carbons, then the three LPE, LPS, LPG and LPI species also have saturated acyl chains of 15, 17 and 19 carbons.

[0097] In one embodiment, the lysophospholipid species included in the ULQS include LPC, LPE, LPS, LPG, and LPI, R2 is a saturated acyl chain of 13, 15, 17, 19, or 21 carbons in length, and at least one of R4 (preferably all R4) is deuterium (D). In one aspect of this embodiment, 3-5 species are present for each of LPC, LPE, LPS, LPG, and LPI, and each of the 15-25 species are different. In another aspect of this embodiment, 3-5 species are present for each of LPC, LPE, LPS, LPG, and LPI, and each of the 15-25 individual species are different, and the R2 group is repeated for each of LPC, LPE, LPS, LPG, and LPI. For example, if the three LPC species have saturated acyl chains of 13, 17 and 21 carbons, then the three LPE, LPS, LPG and LPI species also have saturated acyl chains of 13, 17 and 21 carbons.

[0098] In one embodiment, the lysophospholipid species included in the ULQS are represented by the following formulas IIB-IIF:

[0099] [ka]

[0100] where at least one of R4 (preferably all of R4) is deuterium and R2 is represented by three different saturated acyl chains: i) 13, 15, and 17 carbons in length; ii) 15, 17, and 19 carbons in length; or iii) 17, 19, or 21 carbons in length.

[0101] In specific embodiments, lysophospholipid species of formulae IIA-F included in the ULQS include those listed in Table 2 below, where at least one of R4 (preferably all of R4) is deuterium, LPC is a compound of formula A, LPE is a compound of formula B, LPS is a compound of formula C, LPG is a compound of formula D, and LPI is a compound of formula E, and the underlined portions correspond to the R2 variables.

[0102] [Table 2]

[0103] Cholesterol esters In one embodiment, the cholesterol ester class comprises a plurality of cholesterol ester species, each cholesterol ester species comprises an isotopic label. Any combination of cholesterol ester species described herein can be used in the ULQS. In one embodiment, the cholesterol ester class comprises a plurality of cholesterol ester species, each cholesterol ester species comprises at least one isotopically labeled H atom, preferably 2 to 9 isotopically labeled H atoms. In a preferred embodiment, the isotopically labeled H atom is deuterium (D).

[0104] In one embodiment, cholesterol esters for inclusion in the ULQS have the general formula III:

[0105] [ka]

[0106] or a pharma- ceutically acceptable salt thereof, wherein R2 is a C9 to C29 saturated or unsaturated acyl chain; and R4 is independently H or an isotope of H, provided that at least one of R4 is an isotope of H.

[0107] R2 is a saturated or unsaturated acyl chain between 2 and 29 carbons in length. In one embodiment, R2 is an acyl chain between 2 and 5 carbons in length, an acyl chain between 6 and 10 carbons in length, an acyl chain between 10 and 20 carbons in length, or an acyl chain between 21 and 29 carbons in length. In certain aspects, R2 is an acyl chain between 10 and 20 or 21 and 29 carbons in length. Such acyl chains, regardless of length, include both even and odd chain lengths and can be saturated or unsaturated. In certain embodiments, the acyl chains of the cholesterol ester species are each selected such that the total composition score of the acyl chain is an even number. In another embodiment, R2 is a 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 carbon acyl chain that is saturated or unsaturated. In another embodiment, R2 is a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon acyl chain that is saturated or unsaturated. In another embodiment, R2 is a 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon acyl chain that is saturated or unsaturated.

[0108] In one embodiment, R2 is a saturated acyl chain. In another embodiment, R2 is an unsaturated acyl chain. When R2 is an unsaturated acyl chain, the acyl chain can contain 1-6, 1-4, or 1-3 double bonds. In one embodiment, R2 is an unsaturated acyl chain containing 1-4, 1-3, or 1-2 double bonds. In certain embodiments, R2 contains two or more double bonds when the acyl chain length is 18 or more or 20 or more (e.g., containing 2-4 double bonds). The double bonds in such unsaturated acyl chains may be in cis or trans configuration, or may be a mixture of cis and trans configurations when two or more double bonds are present.

[0109] In one embodiment, when multiple species of cholesterol esters are present, the R2 groups on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-4 double bonds). In one embodiment, when multiple species of cholesterol esters are present, the R2 groups on the various species are selected to include only unsaturated acyl chains (e.g., having 1-6 or 1-4 double bonds). In one embodiment, when multiple species of cholesterol esters are present, the R2 groups on the various species are selected to include only unsaturated acyl chains (e.g., having 1-4 double bonds), including acyl chains with 2-4 double bonds.

[0110] In one embodiment of any of the foregoing, the isotopically labeled H is deuterium (D). In one embodiment of any of the foregoing, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all nine R4 are isotopically labeled H, preferably deuterium (D).

[0111] In some embodiments, at least a portion of the cholesterol ester species in the ULQS have different R2 groups. In some embodiments, all cholesterol ester species in the ULQS have different R2 groups, where the R2 groups are unsaturated acyl chains (e.g., having 1-6 or 1-4 double bonds). In some embodiments, all cholesterol ester species in the ULQS have different R2 groups, where the R2 groups are unsaturated acyl chains (e.g., having 1-6 or 1-4 double bonds), including acyl chains with 2-4 double bonds (e.g., where the acyl chain length is 20 or more carbons).

[0112] For example, if the ULQS includes five cholesterol ester species, the species can be represented by Formula III, where R2 is a C10-C20 unsaturated acyl chain and each of R4 is deuterium.

[0113] In specific embodiments, cholesterol ester species suitable for inclusion in the ULQS include those listed in Table 3 below (with reference to Formula III, where at least one, and preferably all, of R4 are deuterium and the underlined portions correspond to the R2 variables):

[0114] [Table 3]

[0115] Triacylglycerol In one embodiment, the triacylglycerol class comprises a plurality of triacylglycerol species, where each triacylglycerol species comprises an isotopic label. Any combination of triacylglycerol species described herein can be used in the ULQS. In one embodiment, the acyl chains of the triacylglycerol species are independently selected from saturated C3-C30 acyl chains or unsaturated C3-C30 acyl chains containing 1-6 double bonds, such as 1-4 or 1-2 double bonds.

[0116] In one embodiment, the acyl chains of the triacylglycerol species are each independently a saturated or unsaturated C3-C5 acyl chain, a saturated or unsaturated C6-C10 acyl chain, a saturated or unsaturated C11-C20 acyl chain, and a saturated or unsaturated C21-C30 acyl chain. Such acyl chains, regardless of length, include both even and odd chain lengths and can be saturated or unsaturated. In certain embodiments, the acyl chains of the triacylglycerol species are each selected such that the total composition score of the acyl chains is an odd number. In certain embodiments, the acyl chains of the triacylglycerol species are each a 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon acyl chain that is saturated or unsaturated. In another specific embodiment, the acyl chains of the triacylglycerol species are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 acyl chains, each of which is independently saturated or unsaturated. In yet another specific embodiment, the acyl chains of the triacylglycerol species are 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 acyl chains, each of which is independently saturated or unsaturated carbons.

[0117] In one embodiment, one or more of the acyl chains of the triacylglycerol species are saturated. In another embodiment, one or more of the acyl chains of the triacylglycerol species are unsaturated. In yet another embodiment, one of the acyl chains is saturated and two of the acyl chains are unsaturated. In yet another embodiment, two of the acyl chains are saturated and one of the acyl chains is unsaturated. In yet another embodiment, all three acyl chains are unsaturated. When one or more of the acyl chains of the triacylglycerol species are unsaturated, the acyl chains can contain 1-6, 1-4, 1-3, or 1-2 double bonds. In one embodiment, at least one, two, or all three of the acyl chains are unsaturated and contain 1-4, 1-3, or 1-2 double bonds. In some embodiments, at least one of the acyl chains contains two or more double bonds when the acyl chain length is greater than 18 (including, for example, two double bonds). The double bonds in such unsaturated acyl chains may be in the cis or trans configuration, or if more than one double bond is present, may be a mixture of cis and trans configurations.

[0118] In one embodiment, when multiple species of a particular triacylglycerol are present, the acyl chains on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-4 double bonds), or the acyl chains on the various species are selected to include only unsaturated acyl chains (e.g., having 1-4 double bonds). In one embodiment, when multiple species of triacylglycerol are present, the acyl chains on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1 and 2 double bonds), including acyl chains with 2 double bonds when the acyl chain length is greater than 18. In one embodiment, when multiple species of triacylglycerol are present, the acyl chains on the various species are selected to include only unsaturated acyl chains (e.g., having 1 and 2 double bonds), including acyl chains with 2 double bonds when the acyl chain length is greater than 18.

[0119] In some embodiments, when multiple triacylglycerol species are present, the length of one acyl chain is different from the length of the other two acyl chains (preferably the same). In some embodiments, when multiple triacylglycerol species are present, the length of one acyl chain is different from the length of the other two acyl chains (preferably the same), and at least one acyl chain is unsaturated (e.g., contains 1-4 double bonds) and at least one acyl chain is saturated (no double bonds). In some embodiments, when multiple triacylglycerol species are present, the length of one acyl chain is different from the length of the other two acyl chains (preferably the same), and each of the acyl chains of the same length is saturated and the acyl chains of different lengths are unsaturated (e.g., contains 1-2 double bonds and 2 double bonds when the acyl chain length is greater than 18 carbons). In some embodiments, when multiple triacylglycerol species are present, the length of one acyl chain is different from the length of the other two acyl chains (preferably the same), and each acyl chain is unsaturated.

[0120] In some embodiments, when multiple triacylglycerol species are present, the total composition scores of the different species are different from each other. In some embodiments, when multiple triacylglycerol species are present, the total composition scores of the different species are different from each other, and one or two of the acyl chains are unsaturated (e.g., containing 1-4 double bonds, particularly 2-3 double bonds when the acyl chain length is greater than 18), and one or two acyl chains are saturated. In one embodiment, the total composition scores of the acyl chains of the different species are odd numbers.

[0121] In any of the foregoing embodiments, the triacylglycerol comprises at least one isotopically labeled H atom, preferably 2 to 5 isotopically labeled H atoms. In a preferred embodiment, the isotopically labeled H atom is deuterium (D).

[0122] In one such embodiment, triacylglycerols for inclusion in the ULQS have the general formula IV:

[0123] [ka]

[0124] or a pharma- ceutically acceptable salt thereof, wherein R2 is a C3-C30 saturated or unsaturated acyl chain; R3 are each independently a C3 to C25 saturated or unsaturated acyl chain; R4 is independently H or an isotope of H, provided that at least one of R4 is an isotope of H.

[0125] R2 is a saturated or unsaturated acyl chain between 3 and 30 carbons in length. In one embodiment, R2 is an acyl chain between 3 and 5 carbons in length, an acyl chain between 6 and 10 carbons in length, an acyl chain between 10 and 20 carbons in length, or an acyl chain between 21 and 30 carbons in length. In a particular embodiment, R2 is an acyl chain between 10 and 25 carbons in length. In another particular embodiment, R2 is an acyl chain between 12 and 23 carbons in length. Such acyl chains, regardless of length, include both even and odd chain lengths and can be saturated or unsaturated. In a particular embodiment, R2 is an acyl chain having an odd number of carbon atoms. In another embodiment, R2 is a 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon acyl chain that is saturated or unsaturated. In another embodiment, R2 is a 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 carbon acyl chain that is saturated or unsaturated. In another embodiment, R2 is a 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon acyl chain that is saturated or unsaturated. In a preferred embodiment, the acyl chain of the above length is unsaturated with 1 to 6, for example 1 to 4, or 1 to 2 double bonds.

[0126] In one embodiment, R2 is a saturated acyl chain. In another embodiment, R2 is an unsaturated acyl chain. When R2 is an unsaturated acyl chain, the acyl chain can contain 1-6, 1-4, or 1-3 double bonds. In one embodiment, R2 is an unsaturated acyl chain containing 1-4, 1-3, or 1-2 double bonds. In certain embodiments, R2 contains 2-4 double bonds (e.g., 2 double bonds) when the acyl chain length is greater than 18. The double bonds in such unsaturated acyl chains may be in cis or trans configuration, or may be a mixture of cis and trans configurations when more than one double bond is present.

[0127] In one embodiment, when multiple triacylglycerol species are present, the R2 groups on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-4 double bonds). In one embodiment, when multiple triacylglycerol species are present, the R2 groups on the various species are selected to include only unsaturated acyl chains (e.g., having 1-4 double bonds). In one embodiment, when multiple triacylglycerol species are present, the R2 groups on the various species are selected to include only unsaturated acyl chains (e.g., having 1-4 double bonds), with acyl chains having 2-4 double bonds, preferably 2 double bonds, when the acyl chain length is longer than 18 carbons.

[0128] R3 is a saturated or unsaturated acyl chain 3-25 carbons in length. In one embodiment, R3 is an acyl chain 3-5 carbons in length, an acyl chain 6-10 carbons in length, an acyl chain 10-20 carbons in length, or an acyl chain 21-25 carbons in length. In a particular embodiment, R3 is an acyl chain 10-20 carbons in length. In another particular embodiment, R3 is an acyl chain 12-20 carbons in length. This includes both even and odd chain lengths and may be saturated or unsaturated. In a particular embodiment, R3 is an acyl chain having an even number of carbon atoms. In another embodiment, R3 is a 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon acyl chain that is saturated or unsaturated. In another embodiment, R3 is a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon acyl chain that is saturated or unsaturated. In another embodiment, R3 is a 14, 15, 16, 17, or 18 carbon acyl chain that is saturated or unsaturated. In a preferred embodiment, the acyl chains of the aforementioned lengths are saturated if the acyl chain is 14 carbons or less and unsaturated if the acyl chain is greater than 15 carbons (e.g., containing 1-4 double bonds).

[0129] In one embodiment, R3 is a saturated acyl chain. In another embodiment, R3 is an unsaturated acyl chain. When R3 is an unsaturated acyl chain, the acyl chain can contain 1-4 double bonds, e.g., 1-2 double bonds. In one embodiment, R3 is an unsaturated acyl chain containing 1-2 double bonds (e.g., when the acyl chain is greater than 17 carbons in length). In one embodiment, R3 is an unsaturated acyl chain containing 1 double bond when the acyl chain is greater than 17 carbons in length. The double bonds in such unsaturated acyl chains may be in the cis or trans configuration, or may be a mixture of cis and trans configurations when more than one double bond is present.

[0130] In one embodiment, when multiple triacylglycerol species are present, the R3 groups on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-2 double bonds).In one embodiment, when multiple triacylglycerol species are present, the R3 groups on the various species are selected to include only saturated acyl chains.

[0131] In one embodiment of any of the foregoing, the isotopically labeled H is deuterium (D). In one embodiment of any of the foregoing, at least one, at least two, at least three, at least four, or all five of R4 are isotopically labeled H, preferably deuterium (D).

[0132] In some embodiments, at least a portion of the triacylglycerol species in the ULQS have the same R3 group and different R2 groups. In some embodiments, all of the triacylglycerol species in the ULQS have the same R3 group and different R2 groups, where the R3 group is a saturated or unsaturated acyl chain (e.g., having 1-2 double bonds) and the R2 group is a saturated or unsaturated acyl chain (e.g., having 1-4 double bonds). In some aspects, all of the triacylglycerol species in the ULQS have the same R3 group and different R2 groups, where the R3 group is a saturated or unsaturated acyl chain (e.g., having 1-2 double bonds) and the R2 group is a saturated or unsaturated acyl chain (e.g., having 1-4 double bonds), including acyl chains with 2-3 double bonds when the acyl chain length is greater than 18. In some embodiments, all of the triacylglycerol species included in the ULQS have the same R3 group and different R2 groups, where the R3 group is saturated when the acyl chain length is less than 18, unsaturated (e.g., having 1-2 double bonds) when the acyl chain length is 18 or greater, and the R2 group is saturated when the acyl chain length is less than 15 or unsaturated (e.g., having 1-4 double bonds) when the acyl chain length is 15 or greater, and includes an acyl chain with 2 double bonds when the acyl chain length is greater than 18.

[0133] In one embodiment, triacylglycerol species included in the ULQS include species where each R3 is an acyl chain of n atoms in length, where n is 10 to 22, and R2 is an acyl chain of n-3 to n+5 carbon atoms, where R3 is saturated or unsaturated, and where R2 is n-3 to n+1 carbon atoms in length, R2 is saturated or unsaturated, and where R2 is n+2 to n+5 carbon atoms in length, R2 contains 2 or 3 double bonds (preferably 2 double bonds).

[0134] In one embodiment, triacylglycerol species included in the ULQS include species in which R3 is independently an acyl chain of n atoms of length n, where n is 12, 14, 16, 18, 20, or 22, R2 is an acyl chain of n-3 to n+5 carbon atoms, where R3 is saturated when n is 12, 14, or 16, and unsaturated (containing 1 to 2 double bonds, preferably 1 double bond) when n is 18, 20, or 22, where R2 contains 1 or 2 double bonds when R2 is n-3 to n+1 carbon atoms in length, and where R2 contains 2 or 3 double bonds (preferably 2 double bonds) when R2 is n+2 to n+5 carbon atoms in length.

[0135] In one embodiment, triacylglycerol species suitable for inclusion in a ULQS include those in which R3 is independently an acyl chain of n carbon atoms in length, where n is 14, 16, or 18; R2 is an acyl chain of n-3 to n+5 carbon atoms, where R3 is saturated when n is 14 or 16, and unsaturated (containing 1-2 double bonds, preferably 1 double bond) when n is 18; When R2 is from n-3 to n+1 carbon atoms in length, R2 includes species that contain 1 or 2 double bonds, and when R2 is from n+2 to n+5 carbon atoms in length, R2 includes species that contain 2 or 3 double bonds (preferably 2 double bonds).

[0136] In one embodiment, a triacylglycerol species suitable for inclusion in a ULQS can be represented by formula IV, wherein: At least one of R4 (preferably all of R4) is deuterium; R3 is a saturated acyl chain of 12, 14, or 16 carbons, or an unsaturated acyl chain of 18 or 20 carbons containing one double bond, respectively; R2 is a saturated or unsaturated acyl chain of 11, 13, 15, 17, 19, 21, 22, or 23 carbons in length, where R2 is saturated when it is 11 or 13 carbons in length, R2 contains one double bond when it is 15 or 17 carbons in length, and R2 contains two double bonds when it is 19, 21, or 23 carbons in length.

[0137] In certain aspects of this embodiment, the triacylglycerol species suitable for inclusion in the ULQS consist of nine species, where R3 is 12:0 and R2 is 11:0, 13:1 and 15:1, R3 is 14:0 and R2 is 13:0, 15:1 and 17:1, R3 is 16:0 and R2 is 15:1, 17:1 and 19:2.

[0138] The cerol species consists of nine species, where R3 is 14:0 and R2 are 13:0, 15:1 and 17:1, R3 is 16:0 and R2 are 15:1, 17:1 and 19:2, R3 is 18:1 and R2 are 17:1, 19:2 and 21:2.

[0139] In particular aspects of this embodiment, the triacylglycerol species suitable for inclusion in the ULQS consist of nine species, where R3 is 16:0 and R2 is 15:1, 17:1 and 19:2, R3 is 18:1 and R2 is 17:1, 19:2 and 21:2, R3 is 20:1 and R2 is 19:2, 21:2 and 23:2.

[0140] In certain embodiments, triacylglycerol species suitable for inclusion in the ULQS include those listed in Table 4 below (with reference to Formula IV, at least one of R4 (preferably all of R4) is deuterium, the underlined portions correspond to the R2 variables, and the double underlined portions correspond to the R3 variables).

[0141] [Table 4]

[0142] Diacylglycerol In one embodiment, the diacylglycerols include a plurality of diacylglycerol species, where each diacylglycerol species includes an isotopic label. Any combination of diacylglycerol molecules known in the art can be used. In one embodiment, the acyl chains of the diacylglycerol species are independently selected from saturated C3-C30 acyl chains or unsaturated C3-C30 acyl chains containing 1-6 double bonds, such as 1-4 or 1-2 double bonds.

[0143] In one embodiment, both of the acyl chains of the diacylglycerol species are each independently a saturated or unsaturated C3-C6 acyl chain, a saturated or unsaturated C7-C11 acyl chain, a saturated or unsaturated C12-C21 acyl chain, or a saturated or unsaturated C22-C30 acyl chain. Such acyl chains can be saturated or unsaturated, regardless of length, including both even and odd chain lengths. In certain embodiments, the acyl chains of the diacylglycerol species are each selected such that the total composition score of the acyl chains is an odd number. In certain embodiments, the acyl chains of the diacylglycerol species are each a 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon acyl chain, each of which is saturated or unsaturated. In another particular embodiment, both of the acyl chains of the diacylglycerol species are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon acyl chains that are each saturated or unsaturated. In yet another particular embodiment, both of the acyl chains of the diacylglycerol species are 15, 16, 17, 18, 19, 20, 21, 22, or 23 carbon acyl chains that are each independently saturated or unsaturated.

[0144] In one embodiment, one or both of the acyl chains of the diacylglycerol species are saturated. In another embodiment, one or both of the acyl chains of the diacylglycerol species are unsaturated. In yet another embodiment, one of the acyl chains is saturated and one of the acyl chains is unsaturated. When one or both of the acyl chains of the diacylglycerol species are unsaturated, the acyl chains can contain 1-6, 1-4, 1-3, or 1-2 double bonds. In one embodiment, at least one of the acyl chains is unsaturated and contains 1-6, 1-4, 1-3, or 1-2 double bonds. In some aspects, at least one of the acyl chains contains two or more double bonds (e.g., contains 3-4 double bonds) when the acyl chain length is greater than 18. The double bonds in such unsaturated acyl chains may be in the cis or trans configuration, or may be a mixture of cis and trans configurations when more than one double bond is present.

[0145] In one embodiment, when multiple diacylglycerol species are present, the acyl chains on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-4 double bonds). In one embodiment, when multiple diacylglycerol species are present, the acyl chains on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-4 double bonds), including acyl chains with 3-4 double bonds (preferably when the acyl chain length is greater than 18).

[0146] In some embodiments, when multiple diacylglycerol species are present, the length of one acyl chain is different from the length of the other acyl chain. In some embodiments, when multiple diacylglycerol species are present, the length of one acyl chain is different from the length of the other acyl chain, and one acyl chain is unsaturated (e.g., contains 1-4 double bonds) and one acyl chain is saturated (no double bonds). In certain embodiments, when multiple diacylglycerol species are present, the length of one acyl chain is different from the length of the other acyl chain, and one acyl chain is unsaturated and contains one double bond when the acyl chain length is greater than 18, shorter than 18 and 3-4 double bonds.

[0147] In some embodiments, when multiple diacylglycerol species are present, the total composition scores of the different species are different from each other. In certain embodiments, when multiple diacylglycerol species are present, the total composition scores of the different species are different from each other, and one of the acyl chains is unsaturated (e.g., contains 1-4 double bonds, particularly 3-4 double bonds when the acyl chain length is greater than 18) and the other acyl chain is saturated. In one embodiment, the total composition scores of the acyl chains of the different species are odd numbers.

[0148] In any of the foregoing embodiments, the diacylglycerol comprises at least one isotopically labeled H atom, preferably 2 to 5 isotopically labeled H atoms. In a preferred embodiment, the isotopically labeled H atom is deuterium (D).

[0149] In one such embodiment, diacylglycerols for inclusion in the ULQS have the general formula V:

[0150] [ka]

[0151] In the formula: R2 is a C3-C30 saturated or unsaturated acyl chain; R3 is a C3-C25 saturated or unsaturated acyl chain; R4 is independently H or an isotope of H, provided that at least one of R4 is an isotope of H.

[0152] R2 is a saturated or unsaturated acyl chain between 3 and 30 carbons in length. In one embodiment, R2 is an acyl chain between 3 and 6 carbons in length, an acyl chain between 7 and 11 carbons in length, an acyl chain between 12 and 21 carbons in length, or an acyl chain between 22 and 30 carbons in length. In a particular embodiment, R2 is an acyl chain between 10 and 20 or between 21 and 25 carbons in length. In another particular embodiment, R2 is an acyl chain between 12 and 24 carbons in length. Such acyl chains, regardless of length, include both even and odd chain lengths and may be saturated or unsaturated. In a particular embodiment, R2 is an acyl chain having an even number of carbon atoms. In another embodiment, R2 is a 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon acyl chain that is saturated or unsaturated. In another embodiment, R2 is a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon acyl chain that is saturated or unsaturated. In another embodiment, R2 is a 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon acyl chain that is saturated or unsaturated. In a preferred embodiment, the acyl chain of the above length is unsaturated with 1 to 6, for example 1 to 4, or 1 to 2 double bonds. In a preferred embodiment, the acyl chain of the above length is unsaturated, containing one double bond when the acyl chain length is less than 18, and containing 3 to 4 double bonds when the acyl chain length is more than 18.

[0153] In one embodiment, R2 is a saturated acyl chain. In another embodiment, R2 is an unsaturated acyl chain. When R2 is an unsaturated acyl chain, the acyl chain can include 1-6, 1-4, or 1-3 double bonds. In one embodiment, R2 is an unsaturated acyl chain including 1-4, 1-3, or 1-2 double bonds. In some embodiments, R2 includes 3 double bonds when the acyl chain length is greater than 18 (including 2 or more double bonds, e.g., 2-4 double bonds). In certain embodiments, R2 includes 3 double bonds when the acyl chain length is greater than 19. In certain embodiments, R2 includes 4 double bonds when the acyl chain length is greater than 21. The double bonds in such unsaturated acyl chains may be in the cis or trans configuration, or may be a mixture of cis and trans configurations when more than one double bond is present.

[0154] In one embodiment, when multiple diacylglycerol species are present, the R2 groups on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-4 double bonds). In one embodiment, when multiple diacylglycerol species are present, the R2 groups on the various species are selected to include only unsaturated acyl chains (e.g., having 1-4 double bonds). In one embodiment, when multiple diacylglycerol species are present, the R2 groups on the various species are selected to include only unsaturated acyl chains (e.g., having 1-4 double bonds), including acyl chains with 2-4 double bonds. In certain embodiments, when the acyl chain length is greater than 19, R2 includes 3 double bonds. In certain embodiments, when the acyl chain length is greater than 21, R2 includes 4 double bonds.

[0155] In certain embodiments, the acyl chain length of R2 is different from the acyl chain length of R3. In certain embodiments, the acyl chain length of R2 is different from the acyl chain length of R3, and R2 is unsaturated (e.g., contains 1-4 double bonds) and R3 is saturated (no double bonds). In certain embodiments, R2 contains 3 double bonds when the acyl chain length is 19 or more (e.g., contains 3-4 double bonds) and R3 is saturated. In certain embodiments, R2 contains 4 double bonds when the acyl chain length is greater than 21 and R3 is saturated.

[0156] R3 is a saturated or unsaturated acyl chain 3-25 carbons in length. In one embodiment, R3 is an acyl chain 3-5 carbons in length, an acyl chain 6-10 carbons in length, an acyl chain 10-20 carbons in length, or an acyl chain 21-25 carbons in length. In a particular embodiment, R3 is an acyl chain 10-20 carbons in length. In another particular embodiment, R3 is an acyl chain 15-19 carbons in length. Such acyl chains, regardless of length, include both even and odd chain lengths and can be saturated or unsaturated. In a particular embodiment, R3 is an acyl chain having an odd number of carbon atoms. In another embodiment, R3 is a 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon acyl chain that is saturated or unsaturated. In another embodiment, R3 is a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon acyl chain that is saturated or unsaturated. In another embodiment, R3 is a 14, 15, 16, 17, or 18 carbon acyl chain that is saturated or unsaturated. In a preferred embodiment, the acyl chains of the aforementioned lengths are saturated.

[0157] In one embodiment, R3 is a saturated acyl chain. In another embodiment, R3 is an unsaturated acyl chain. When R3 is an unsaturated acyl chain, the acyl chain can contain 1 to 4, for example 1 to 2, double bonds. In one embodiment, R3 is an unsaturated acyl chain containing one double bond. The double bonds in such unsaturated acyl chains can be in cis or trans configuration, or can be a mixture of cis and trans configurations when more than one double bond is present.

[0158] In one embodiment, when multiple diacylglycerol species are present, the R3 groups on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-2 double bonds).In one embodiment, when multiple diacylglycerol species are present, the R3 groups on the various species are selected to include only saturated acyl chains.

[0159] In certain embodiments, the acyl chain length of R3 is different from the acyl chain length of R2. In certain embodiments, the acyl chain length of R3 is different from the acyl chain length of R2, and R3 is saturated and R2 is unsaturated (e.g., contains 1-4 double bonds). In certain embodiments, when the acyl chain length is greater than 19 and R3 is saturated, R2 contains 3 double bonds. In certain embodiments, when the acyl chain length is greater than 21 and R3 is saturated, R2 contains 4 double bonds.

[0160] In one embodiment of any of the foregoing, the isotopically labeled H is deuterium (D). In one embodiment of any of the foregoing, at least one, at least two, at least three, at least four, or all five of R4 are isotopically labeled H, preferably deuterium (D).

[0161] In some embodiments, at least a portion of the diacylglycerol species in the ULQS have the same R3 group and different R2 groups. In some embodiments, all of the diacylglycerol species in the ULQS have the same R3 group and different R2 groups, where the R2 group is an unsaturated acyl chain (e.g., having 1-4 double bonds). In some embodiments, all of the diacylglycerol species in the ULQS have the same R3 group and different R2 groups, where the R3 group is a saturated acyl chain and the R2 group is an unsaturated acyl chain (e.g., having 1-4 double bonds). In some embodiments, all of the diacylglycerol species in the ULQS have the same R3 group and different R2 groups, where the R2 group is an unsaturated acyl chain (e.g., having 1-4 double bonds), including an acyl chain with 2-4 double bonds. In certain embodiments, all of the diacylglycerol species included in the ULQS have the same R3 group and different R2 groups, where the R3 group is a saturated acyl chain and the R2 group is an unsaturated acyl chain (e.g., having 1-4 double bonds), including acyl chains having 3-4 double bonds. In certain of the foregoing embodiments, R2 includes 3 double bonds when the acyl chain length is greater than 21, when the acyl chain length is 19 and greater than 4 double bonds.

[0162] In one embodiment, diacylglycerol species included in the ULQS include species where R3 is independently an acyl chain of length n carbon atoms, where n is 14 to 20, and R2 is independently an acyl chain of n-5 to n+7 carbon atoms, where R3 optionally contains 1 or 2 double bonds (preferably 0 double bonds), where R2 is n-5 to n-1 carbon atoms in length, where R2 contains 1 or 2 double bonds (preferably 1 double bond), and where R2 is n+2 to n+7 carbon atoms in length, where R2 contains 2 to 4 double bonds (preferably 3 or 4 double bonds).

[0163] In one embodiment, diacylglycerol species included in the ULQS include species where R3 is independently an acyl chain of length n carbon atoms, where n is 15, 17, or 19 and contains double bonds (preferably 0 double bonds), where R2 contains 1 or 2 double bonds (preferably 1 double bond) when R2 is n-5 to n-1 carbon atoms in length, and where R2 contains 2 to 4 double bonds (preferably 3 or 4 double bonds) when R2 is n+2 to n+7 carbon atoms in length.

[0164] In one embodiment, diacylglycerol species included in the ULQS include species where R3 is independently a saturated acyl chain of length n, where n is 15, 17, or 19, R2 is independently an acyl chain of n-5 to n+7 carbon atoms, R3 optionally containing 1 or 2 double bonds (preferably 0 double bonds), when R2 is n-5 to n-1 carbon atoms in length, R2 contains 1 or 2 double bonds (preferably 1 double bond), when R2 is n+2 to n+3 carbon atoms in length, R2 contains 2 to 3 double bonds (preferably 3 double bonds), and when R2 is n+4 to n+7 carbon atoms in length, R2 contains 3 to 4 double bonds (preferably 4 double bonds).

[0165] In one embodiment, a diacylglycerol species suitable for inclusion in a ULQS can be represented by formula V, wherein: At least one of R4 (preferably all of R4) is deuterium; R3 is a saturated acyl chain of 15, 17, or 19 carbons in length; R2 is an acyl chain of 12, 14, 16, 18, 20, 22, or 24 carbons in length, where when R2 is 12, 14, 16, or 18 carbons in length, R2 contains one double bond, when R2 is 20 carbons in length, R2 contains three double bonds, and when R2 is 22 or 24 carbons in length, R2 contains four double bonds.

[0166] In certain aspects of this embodiment, the diacylglycerol species contained in the ULQS consists of five species, where R3 is 15 carbons in length and R2 are saturated acyl chains of 14:1, 16:1, 18:1, 20:3, and 22:4.

[0167] In another particular aspect of this embodiment, the diacylglycerol species contained in the ULQS consists of five species, where R3 is a saturated acyl chain 17 carbons in length and R2 is a saturated acyl chain of 14:1, 16:1, 18:1, 20:3, and 22:4.

[0168] In another particular aspect of this embodiment, the ULQS comprises five diacylglycerol species, where R3 is 19 carbons in length and R2 are saturated acyl chains of 14:1, 16:1, 18:1, 20:3, and 22:4.

[0169] In certain embodiments, diacylglycerol species suitable for inclusion in the ULQS include those listed in Table 5 below (with reference to Formula V, where at least one of R4 (preferably all of R4) is deuterium, the underlined portions correspond to the R2 variables and the double underlined portions correspond to the R3 variables).

[0170] [Table 5]

[0171] Ceramide In one embodiment, the ceramides include a plurality of ceramide species, where each ceramide species includes an isotopic label. Any combination of ceramides described herein can be used. In one embodiment, the acyl chains of the ceramide species are independently selected from saturated C10-C30 acyl chains or unsaturated C10-C30 acyl chains containing 1-4 double bonds, e.g., 1-2 double bonds or 1 double bond.

[0172] In one embodiment, the acyl chain of the ceramide species is a saturated or unsaturated C10-C15 acyl chain, a saturated or unsaturated C16-C20 acyl chain, a saturated or unsaturated C15-C25 acyl chain, a saturated or unsaturated C21-C25 acyl chain, or a saturated or unsaturated C26-C30 acyl chain. Such acyl chains can be saturated or unsaturated, regardless of length, including both even and odd chain lengths. In certain embodiments, the acyl chain of the ceramide species is selected such that the total composition score of the acyl chain is an even number. In certain embodiments, the acyl chain of the ceramide species is a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon acyl chain, each of which is saturated or unsaturated (preferably unsaturated). In another specific embodiment, the acyl chains of the ceramide species are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon acyl chains, each of which is independently saturated or unsaturated (preferably unsaturated). In yet another specific embodiment, the acyl chains of the ceramide species are 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon acyl chains, each of which is independently saturated or unsaturated (preferably unsaturated).

[0173] In one embodiment, the acyl chain of the ceramide species is saturated. In another embodiment, the acyl chain of the ceramide species is unsaturated. When the acyl chain of the ceramide species is unsaturated, the acyl chain may contain 1 to 4 double bonds. In one embodiment, the acyl chain is unsaturated containing 1 or 2 double bonds. The double bonds in such unsaturated acyl chains may be in the cis or trans configuration, or may be a mixture of cis and trans configurations when more than one double bond is present.

[0174] In one embodiment, when multiple species of ceramides are present, the acyl chains on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-4 double bonds, preferably 1-2 double bonds). In one embodiment, when multiple species of ceramides are present, the acyl chains on the various species are selected to include only unsaturated acyl chains (e.g., having 1-4 double bonds, preferably 1-2 double bonds).

[0175] In some embodiments, when multiple types of ceramides are present, the total composition scores of the acyl chains of the ceramide species are different. In some embodiments, when multiple types of ceramides are present, the total composition is such that the acyl chains of the ceramide species are different, and the acyl chains are each unsaturated (e.g., containing 1-4 double bonds, preferably 1-2 double bonds). In certain embodiments, when multiple types of ceramides are present, the total composition scores of the acyl chains of the ceramide species are different, and the acyl chains are each unsaturated (e.g., containing 1-4 double bonds, preferably 1-2 double bonds), and the total composition score of the acyl chains is an even number.

[0176] In any of the foregoing embodiments, the ceramide comprises at least one isotopically labeled H atom, preferably 2 to 7 isotopically labeled H atoms. In a preferred embodiment, the isotopically labeled H atom is deuterium (D).

[0177] In one such embodiment, the ceramide for inclusion in the ULQS has the general formula VI:

[0178] [ka]

[0179] or a pharma- ceutically acceptable salt thereof, wherein R2 is a C10-C30 saturated or unsaturated acyl chain; R4 is independently H or an isotope of H, provided that at least one of R4 is an isotope of H.

[0180] R2 is a saturated or unsaturated acyl chain having 10-30 carbons. In one embodiment, R2 is an acyl chain having a length of 10-15 carbons, an acyl chain having a length of 16-20 carbons, an acyl chain having a length of 21-25 carbons, or an acyl chain having a length of 26-30 carbons. In a particular embodiment, R2 is an acyl chain having a length of 10-25 carbons. In another particular embodiment, R2 is an acyl chain having a length of 16-24 carbons. Such acyl chains may be saturated or unsaturated, regardless of length, including both even and odd chain lengths. In a particular embodiment, R2 is an acyl chain having an even number of carbon atoms. In another embodiment, R2 is a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon acyl chain that is saturated or unsaturated (preferably unsaturated). In another embodiment, R2 is a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon acyl chain that is saturated or unsaturated (preferably unsaturated). In another embodiment, R2 is a 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon acyl chain that is saturated or unsaturated (preferably unsaturated). In a preferred aspect, the acyl chain of the aforementioned length is unsaturated with 1-4 double bonds, preferably 1-2 double bonds.

[0181] In one embodiment, R2 is a saturated acyl chain. In another embodiment, R2 is an unsaturated acyl chain. When R2 is an unsaturated acyl chain, the acyl chain can contain 1-4 double bonds. In one embodiment, R2 is an unsaturated acyl chain containing 1 or 2 double bonds. The double bonds in such unsaturated acyl chains can be in the cis or trans configuration, or can be a mixture of cis and trans configurations when more than one double bond is present.

[0182] In one embodiment, when multiple ceramide species are present, the R2 groups on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-4 double bonds). In one embodiment, when multiple ceramide species are present, the R2 groups on the various species are selected to include only unsaturated acyl chains (e.g., having 1-4 double bonds, preferably 1-2 double bonds).

[0183] In one embodiment of any of the foregoing, the isotopically labeled H is deuterium (D). In one embodiment of any of the foregoing, at least one, at least two, at least three, at least four, at least five, at least six, or all seven of R4 are isotopically labeled H, preferably deuterium (D).

[0184] In some embodiments, at least a portion of the ceramide species in the ULQS have different R2 groups. In some aspects, all of the ceramide species in the ULQS have different R2 groups, and each R2 group is unsaturated (e.g., has 1-2 double bonds).

[0185] In one embodiment, a ceramide species suitable for inclusion in a ULQS can be represented by Formula VI, wherein: At least one of R4 (preferably all of R4) is deuterium; R2 is an acyl chain of 12, 14, 16, 18, 20, 22, 24, 26, or 28 carbons in length, and R2 contains one double bond.

[0186] In a particular aspect of this embodiment, the ceramide species suitable for inclusion in the ULQS consist of five species where R2 is 12:1, 14:1, 16:1, 18:1, and 20:1.

[0187] In a particular aspect of this embodiment, the ceramide species suitable for inclusion in the ULQS consist of five species where R2 is 14:1, 16:1, 18:1, 20:1, and 22:1.

[0188] In a particular aspect of this embodiment, the ceramide species suitable for inclusion in the ULQS consist of five species where R2 is 16:1, 18:1, 20:1, 22:1, and 24:1.

[0189] In a particular aspect of this embodiment, the ceramide species suitable for inclusion in the ULQS consist of five species where R2 is 18:1, 20:1, 22:1, 24:1, and 26:1.

[0190] In a particular aspect of this embodiment, the ceramide species suitable for inclusion in the ULQS consist of five species where R2 is 20:1, 22:1, 24:1, 26:1, and 28:1.

[0191] In specific embodiments, ceramide species suitable for inclusion in the ULQS include those listed in Table 6 below (with reference to Formula VI, where at least one of R4 (preferably all R4) is deuterium and the underlined portions correspond to the R2 variables):

[0192] [Table 6]

[0193] Sphingomyelin In one embodiment, the sphingomyelins include multiple sphingomyelin species. Any combination of sphingomyelin species described herein can be used. In one embodiment, the acyl chains of the sphingomyelin species are independently selected from saturated C10-C30 acyl chains or unsaturated C10-C30 acyl chains containing 1-4 double bonds, e.g., 1-2 double bonds or 1 double bond. In one embodiment, the sphingomyelin species includes a phosphatidylcholine group. In another embodiment, the sphingomyelin species includes a phosphatidylethanolamine group.

[0194] In one embodiment, the acyl chains of the sphingomyelin species are independently saturated or unsaturated C10-C15 acyl chains, saturated or unsaturated C16-C20 acyl chains, saturated or unsaturated C21-C25 acyl chains, or saturated or unsaturated C26-C30 acyl chains. Such acyl chains can be saturated or unsaturated, regardless of length, including both even and odd chain lengths. In certain embodiments, the acyl chains of the sphingomyelin species are each selected such that the total composition score is an even number. In certain embodiments, the acyl chains of the sphingomyelin species are independently 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon acyl chains that are saturated or unsaturated (preferably unsaturated). In another specific embodiment, the acyl chain of the sphingomyelin species is a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon acyl chain that is independently saturated or unsaturated (preferably unsaturated). In yet another specific embodiment, the acyl chain of the sphingomyelin species is a 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon acyl chain that is independently saturated or unsaturated (preferably unsaturated).

[0195] In one embodiment, the acyl chains of the sphingomyelin species are saturated. In another embodiment, the acyl chains of the sphingomyelin species are unsaturated. When one or more of the acyl chains of the sphingomyelin species are unsaturated, the acyl chains can contain 1 to 4 double bonds, preferably 1 to 2 double bonds. In one embodiment, the acyl chains are unsaturated and contain 1 or 2 double bonds, preferably 1 double bond. The double bonds in such unsaturated acyl chains may be in the cis or trans configuration, or may be a mixture of cis and trans configurations when more than one double bond is present.

[0196] In one embodiment, when multiple sphingomyelin species are present, the acyl chains on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-4 double bonds). In one embodiment, when multiple sphingomyelin species are present, the acyl chains on the various species are selected to include only unsaturated acyl chains (e.g., having 1-4 double bonds, preferably 1-2 double bonds, more preferably 1 double bond).

[0197] In certain embodiments, when multiple sphingomyelin species are present, the total composition scores of the different species are different from one another. In certain embodiments, when multiple sphingomyelin species are present, the total composition scores of the different species are different from one another, the acyl chains are each unsaturated (e.g., containing 1-4 double bonds, preferably 1-2 double bonds, more preferably 1 double bond), and the total composition scores of the acyl chains are even numbers.

[0198] In any of the foregoing embodiments, the sphingomyelin comprises at least one isotopically labeled H atom, preferably 2 to 9 isotopically labeled H atoms. In a preferred embodiment, the isotopically labeled H atom is deuterium (D).

[0199] In one such embodiment, sphingomyelin for inclusion in the ULQS has the general formula VII:

[0200] [ka]

[0201] wherein: R1 is,

[0202] [ka]

[0203] and; R2 is a C10-C30 saturated or unsaturated acyl chain; R4 and R4' are each independently H or an isotope of H, provided that at least one of R4 or R4' is an isotope of H.

[0204] In one embodiment, R is

[0205] [ka]

[0206] In one embodiment, R1 is

[0207] [ka]

[0208] and each R4 is H. In another embodiment, R1 is

[0209] [ka]

[0210] R1 is,

[0211] [ka]

[0212] When R4 is, at least one, and preferably all, of R4 is an isotope of H (preferably deuterium, D).

[0213] R2 is a saturated or unsaturated acyl chain between 10 and 30 carbons in length. In one embodiment, R2 is an acyl chain between 10 and 15 carbons in length, an acyl chain between 16 and 20 carbons in length, an acyl chain between 21 and 25 carbons in length, or an acyl chain between 26 and 30 carbons in length. In a particular embodiment, R2 is an acyl chain between 10 and 25 carbons in length. In another particular embodiment, R2 is an acyl chain between 16 and 24 carbons in length. Such acyl chains may be saturated or unsaturated, regardless of length, including both even and odd chain lengths. In a particular embodiment, R2 is an acyl chain having an even number of carbon atoms. In another embodiment, R2 is an acyl chain of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbons that is independently saturated or unsaturated (preferably unsaturated). In another embodiment, R2 is an acyl chain of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbons that is independently saturated or unsaturated. In another embodiment, R2 is an acyl chain of 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbons that is independently saturated or unsaturated (preferably unsaturated). In a preferred embodiment, the acyl chain of the aforementioned length is unsaturated with 1 to 4 double bonds.

[0214] In one embodiment, R2 is a saturated acyl chain. In another embodiment, R2 is an unsaturated acyl chain. When R2 is an unsaturated acyl chain, the acyl chain can contain 1-4 double bonds. In one embodiment, R2 is an unsaturated acyl chain containing 1 or 2 double bonds. The double bonds in such unsaturated acyl chains can be in the cis or trans configuration, or can be a mixture of cis and trans configurations when more than one double bond is present.

[0215] In one embodiment, when multiple sphingomyelin species are present, the R2 groups on the various species are selected to include both saturated and unsaturated acyl chains (e.g., having 1-4 double bonds). In one embodiment, when multiple sphingomyelin species are present, the R2 groups on the various species are selected to include only unsaturated acyl chains (e.g., having 1-4 double bonds).

[0216] In any of the above embodiments, the isotopically labeled H is deuterium (D). In any of the above embodiments, at least one, at least two, at least three, or all four of R4 are isotopically labeled H, preferably deuterium (D). In any of the above embodiments, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all nine of R4' are isotopically labeled H, preferably deuterium (D). In any of the above embodiments, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or nine of R4' are isotopically labeled H, preferably deuterium (D), and at least one, at least two, at least three, or preferably all four of R4 are H.

[0217] In some embodiments, at least a portion of the sphingomyelin species included in the ULQS have the same R1 group and different R2 groups. In some examples, at least a portion of the sphingomyelin species included in the ULQS have the same R1 group, different R2 groups, and each R2 group is unsaturated (e.g., has 1-2 double bonds).

[0218] In one embodiment, a sphingomyelin species suitable for inclusion in a ULQS has the following formula VIIB:

[0219] [ka]

[0220] In the formula: At least one of R4' (preferably all of R4') is deuterium; R2 is an acyl chain of 12, 14, 16, 18, 20, 22, 24, 26, or 28 carbons in length, where R2 contains one double bond.

[0221] In a particular aspect of this embodiment, the sphingomyelin species suitable for inclusion in the ULQS consist of five species where R2 is 12:1, 14:1, 16:1, 18:1, and 20:1.

[0222] In a particular aspect of this embodiment, the sphingomyelin species suitable for inclusion in the ULQS consist of five species where R2 is 14:1, 16:1, 18:1, 20:1, and 22:1.

[0223] In a particular aspect of this embodiment, the sphingomyelin species suitable for inclusion in the ULQS consist of five species where R2 is 16:1, 18:1, 20:1, 22:1, and 24:1.

[0224] In a particular aspect of this embodiment, the sphingomyelin species suitable for inclusion in the ULQS consist of five species where R2 is 18:1, 20:1, 22:1, 24:1, and 26:1.

[0225] In particular aspects of this embodiment, the sphingomyelin species suitable for inclusion in the ULQS consist of five species where R2 is 20:1, 22:1, 24:1, 26:1, and 28:1.

[0226] In one embodiment, a sphingomyelin species suitable for inclusion in a ULQS has the following formula VIIC:

[0227] [ka]

[0228] In the formula: At least one of R4 (preferably all of R4) is deuterium; R2 is an acyl chain of 12, 14, 16, 18, 20, 22, 24, 26, or 28 carbons in length, and R2 contains one double bond.

[0229] In a particular aspect of this embodiment, the sphingomyelin species suitable for inclusion in the ULQS consist of five species where R2 is 12:1, 14:1, 16:1, 18:1, and 20:1.

[0230] In a particular aspect of this embodiment, the sphingomyelin species suitable for inclusion in the ULQS consist of five species where R2 is 14:1, 16:1, 18:1, 20:1, and 22:1.

[0231] In a particular aspect of this embodiment, the sphingomyelin species suitable for inclusion in the ULQS consist of five species where R2 is 16:1, 18:1, 20:1, 22:1, and 24:1.

[0232] In a particular aspect of this embodiment, the sphingomyelin species suitable for inclusion in the ULQS consist of five species where R2 is 18:1, 20:1, 22:1, 24:1, and 26:1.

[0233] In a particular aspect of this embodiment, the sphingomyelin species suitable for inclusion in the ULQS consist of five species where R2 is 20:1, 22:1, 24:1, 26:1, and 28:1.

[0234] In certain embodiments, sphingomyelin species included in the ULQS include those listed in Table 7 below (see Formula VIIB), where at least one of R4' (preferably all of R4') is deuterium and the underlined portions correspond to the R2 variables.

[0235] [Table 7]

[0236] Exemplary Combinations As discussed herein, the ULQS may comprise one or more lipid species from one or more lipid classes described herein. It is understood that the selection of lipid classes to include in the ULQS, the selection of individual lipid species within each lipid class to include in the ULQS, and the concentration of individual lipid species within the ULQS may depend on various factors, such as the sample type to be analyzed or the specific research objective (e.g., identification of lipid biomarkers for heart disease). In one embodiment, the ULQS comprises one or more lipid species from at least three lipid classes. In one embodiment, the ULQS comprises one or more lipid species from at least three lipid classes, and the lipid classes are selected from i) phospholipids; ii) lysophospholipids; iii) cholesterol esters; iv) triacylglycerols; v) diacylglycerols; vi) ceramides; and vii) sphingomyelins.

[0237] In a first combination, the ULQS comprises at least one lipid species from each of the following lipid classes: i) phospholipids; ii) lysophospholipids; iii) cholesterol esters; iv) triacylglycerols; v) diacylglycerols; vi) ceramides; and vii) sphingomyelins.

[0238] In a second combination, the ULQS comprises two or more lipid species from each of the following lipid classes: i) phospholipids; ii) lysophospholipids; iii) cholesterol esters; iv) triacylglycerols; v) diacylglycerols; vi) ceramides; and vii) sphingomyelins.

[0239] In the third combination, the ULQS comprises each of the following lipid classes, with the number of lipid species in each lipid class indicated in parentheses: i) phospholipids (5-40); ii) lysophospholipids (5-25); iii) cholesterol esters (3-15); iv) triacylglycerols (5-20); v) diacylglycerols (3-15); vi) ceramides (3-15); and vii) sphingomyelins (3-15).

[0240] In a fourth combination, the ULQS comprises each of the following lipid classes, with the number of lipid species of each lipid class indicated in parentheses: i) phospholipids (5-40); ii) lysophospholipids (5-25); iii) cholesterol esters (3-15); iv) triacylglycerols (5-20); v) diacylglycerols (3-15); vi) ceramides (3-15); and vii) sphingomyelins (3-15); and the lipid species and lipid species concentrations are selected from Table 8 below.

[0241] In a fifth combination, the ULQS includes each of the following lipid classes with the number of lipid species in each lipid class indicated in parentheses: i) phospholipids (25); ii) lysophospholipids (15); iii) cholesterol esters (5); iv) triacylglycerols (9); v) diacylglycerols (5); vi) ceramides (5); and vii) sphingomyelins (5); and the lipid species and lipid species concentrations are selected from Table 8 below.

[0242] [Table 8-1]

[0243] [Table 8-2]

[0244] [Table 8-3]

[0245] In any of the first to third combinations, the members of each lipid class, optionally with additional lipid species, are selected from the lipid species disclosed in Formulas I to VII herein, and the concentrations of the various lipid species are independently selected from 10 to 500 μg / ml, as described herein.

[0246] In any embodiment of the first to third combinations, the members of each lipid class are selected from the lipid species disclosed in Tables 1 to 7 herein, optionally with additional lipid species, and the concentrations of the various lipid species are independently selected from 10 to 500 μg / ml, as described herein.

[0247] For any of the above phospholipid species, at least one R4 (preferably all R4) is deuterium, and / or the total composition score of the lipid species is as follows: i) phospholipids-27:1 to 43:4 (preferably 31:1 to 39:4); ii) lysophospholipids-11:0 to 23:0 (preferably 15:0 to 19:0); iii) cholesterol esters-11:1 to 25:4 (preferably 14:1 to 22:4); iv) triacylglycerols-43:1 to 55:2 (preferably 47:1 to 51:2); v) diacylglycerols-27:1 to 43:4 (preferably 31:1 to 39:4); vi) ceramides-12:1 to 28:1 (preferably 16:1 to 24:1); and vii) sphingomyelin-12:1 to 28:1 (preferably 16:1 to 24:1).

[0248] In any of the above embodiments, the selection of individual species for inclusion in the ULQS is selected to modify, among other things, at least one of the ionization efficiency, extraction efficiency, and differential fragmentation efficiency of at least one lipid species in the sample to be analyzed or tested.In some embodiments, the individual lipid species selected for inclusion in the ULQS is selected to modify at least one of the different fragmentation efficiency, ionization efficiency, and extraction efficiency (preferably both ionization efficiency and extraction efficiency).In some embodiments, the individual lipid species is selected for inclusion in the ULQS. In certain embodiments, the individual lipid species selected for inclusion in the ULQS are selected to modify at least one of the ionization efficiency, extraction efficiency, and differential fragmentation efficiency of at least one lipid species in the sample being tested, and the various lipid species for inclusion in the ULQS from each class are further selected to i) cover the entire or substantially the entire mass range of at least one lipid species present in the sample, ii) mirror the concentration of at least one lipid species present in the sample, or iii) cover the entire or substantially the entire mass range of at least one lipid species present in the sample and mirror the concentration of at least one lipid species present in the sample.

[0249] In certain embodiments, the individual lipid species selected for inclusion in the ULQS modify the fractionation fragmentation efficiency of at least one lipid species in the sample being analyzed or tested, and at least one ionization efficiency and extraction efficiency (preferably both ionization efficiency and extraction efficiency) of at least one lipid species in the sample being analyzed / tested, and further select different lipid species for inclusion in the ULQS from each class that: i) cover the entire or substantially the entire mass range of at least one lipid species present in the sample; ii) mirror the concentration of at least one lipid species present in the sample; or iii) cover the entire or substantially the entire mass range of at least one lipid species present in the sample and mirror the concentration of at least one lipid species present in the sample.

[0250] packaging The ULQS of the present disclosure can be provided in one or more containers. The interior surface of the container is preferably non-reactive with the components, including the lipid components, of the ULQS. Exemplary containers suitable for use with the ULQS are available from Wheaton.

[0251] The ULQS can be dispersed / dissolved in one or more solvents or powders (e.g., lyophilized powders). Suitable solvents include, but are not limited to, methanol and dichloromethane / methanol (1:1). Other solvents can be used as known in the art. The individual lipid species in the ULQS can be present at the same concentration whether in a single container or multiple containers. Alternatively, one or more of the individual lipid species in the ULQS can be present at different concentrations whether in a single container or multiple containers. Any concentration suitable for the individual lipid species can be used. In one embodiment, the individual lipid species is present at 10-500 μg / ml in a 1 ml volume when dispersed / dissolved in a solvent, or 10-500 μg / ml when provided as a powder.

[0252] The individual lipid species included in the ULQS and the concentration of each lipid species in the ULQS may depend on various factors, such as, but not limited to, the sample type to be analyzed or the specific research purpose (e.g., identifying lipid biomarkers for heart disease). For example, the individual lipid species and the concentration of each lipid species may be optimized for the analysis of plasma samples. For example, when the ULQS is optimized for plasma samples, the ULQS may include PC, PE, PS, PG, and PI, and the concentrations of the five species are PC>PG>PI>PE>PS or PC>PG=PI=PE=PS.

[0253] In one embodiment, the ULQS is provided in a single container and all of the individual lipid species of the ULQS are contained within a single container. The individual lipid species may be present in the same concentration or at different concentrations.

[0254] In another embodiment, the ULQS is provided in more than one container, and each individual lipid species of the ULQS is included in at least two containers. In one aspect of this embodiment, each individual class of lipid compound (e.g., i) phospholipids; ii) lysophospholipids; iii) cholesterol esters; iv) triacylglycerols; v) diacylglycerols; vi) ceramides; and vii) sphingomyelin-derived species is provided in a separate container. The individual lipid species within each class of lipid compounds can be present at the same concentration or at different concentrations. This approach allows the ULQS to be customized, since the concentration of lipid species within each class can be determined by the user based on the factors discussed herein.

[0255] In another aspect of this embodiment, at least one individual class of lipid compounds (e.g., i) phospholipids; ii) lysophospholipids; iii) cholesterol esters; iv) triacylglycerols; v) diacylglycerols; vi) ceramides; and vii) sphingomyelins) are provided in separate containers, and / or at least one lipid species of the lipid classes are provided in separate containers. For example, lysophospholipids, cholesterol esters, triacylglycerols, diacylglycerols, ceramides, and sphingomyelin classes can each be provided in separate containers, and individual lipid species of the phospholipid class can each be provided in separate containers. Individual lipid species within each class of lipid compounds can be present at the same or different concentrations. This approach allows for further customization of the ULQS, since the concentration of lipid species within each class, and the concentration of each lipid species of the phospholipid class, can be determined by the user based on the factors described herein.

[0256] In another aspect of this embodiment, all of the individual lipid species of each class of lipid compound (e.g., i) phospholipids; ii) lysophospholipids; iii) cholesterol esters; iv) triacylglycerols; v) diacylglycerols; vi) ceramides; and vii) sphingomyelins are provided in separate containers. The individual lipid species may be present at the same or different concentrations. This approach allows for further customization of the ULQS, as the concentration of the lipid species may be determined by the user based on the factors discussed herein.

[0257] and vii) sphingomyelins, each lipid class packaged in a separate container. In certain aspects of this embodiment, the members of each lipid class are selected from the lipid species disclosed in Tables 1-7 disclosed herein, or from the lipid species described by Formulas I-VII, as described herein, at a concentration of 10-500 μg / ml, optionally with additional lipid species.

[0258] In another preferred embodiment, the ULQS contains two or more lipid species from three or more of the following lipid classes: i) phospholipids; ii) lysophospholipids; iii) cholesterol esters; iv) triacylglycerols; v) diacylglycerols; vi) ceramides; and vii) sphingomyelins, with each lipid class packaged in a separate container. In certain aspects of this embodiment, the members of each lipid class are selected from the lipid species disclosed in Tables 1-7 disclosed herein, or from the lipid species described by Formulas I-VII, as described herein, at a concentration of 10-500 μg / ml, optionally with additional lipid species.

[0259] In another preferred aspect, the ULQS comprises at least one lipid species from each of the following lipid classes: i) phospholipids; ii) lysophospholipids; iii) cholesterol esters; iv) triacylglycerols; v) diacylglycerols; vi) ceramides; and vii) sphingomyelins, each lipid class packaged in a separate container. In a particular aspect of this embodiment, the members of each lipid class are selected from the lipid species disclosed in Tables 1-7 disclosed herein, or from the lipid species described by Formulas I-VII, as described herein, at a concentration of 10-500 μg / ml, optionally with additional lipid species.

[0260] In another preferred aspect, the ULQS contains two or more lipid species from each of the following lipid classes: i) phospholipids; ii) lysophospholipids; iii) cholesterol esters; iv) triacylglycerols; v) diacylglycerols; vi) ceramides; and vii) sphingomyelins, each lipid class packaged in a separate container. In a particular aspect of this embodiment, the members of each lipid class are selected from the lipid species disclosed in Tables 1-7 disclosed herein, or from the lipid species described by Formulas I-VII, as described herein, at a concentration of 10-500 μg / ml, optionally with additional lipid species.

[0261] In another preferred embodiment, the ULQS comprises each of the following lipid classes, the number of which is indicated in parentheses: i) phospholipids (5-40); ii) lysophospholipids (5-25); iii) cholesterol esters (3-15); iv) triacylglycerols (5-20); v) diacylglycerols (3-15); vi) ceramides (3-15); and vii) sphingomyelins (3-15). Each lipid class is packaged in a separate container, and the lipid species and lipid species concentrations are selected from Table 8 herein, optionally with additional lipid species.

[0262] In another preferred embodiment, the ULQS comprises each of the following lipid classes with the number of lipid species in each lipid class indicated within parentheses: i) phospholipids (25); ii) lysophospholipids (15); iii) cholesterol esters (5); iv) triacylglycerols (9); v) diacylglycerols (5); vi) ceramides (5); and vii) sphingomyelins (5). Each lipid class is packaged in a separate container, and the lipid species and lipid species concentrations are selected from Table 8 herein, optionally with additional lipid species.

[0263] kit The present disclosure also provides a kit for use in quantifying lipid analytes by mass spectrometry.Suitable kits can include the QULS of the present disclosure, and can optionally include packaging materials and instructions for the use of the ULQS.In some embodiments, the ULQS is provided as a packaged reagent set that contains a defined amount of lipid species, and each class of lipid is contained in a separate container in an amount sufficient for at least one assay, as described herein. EXAMPLES

[0264] Example 1 As discussed herein, ULQS can be used in any MS application. A representative MS experiment utilizing ULQS is shown below. In this example, HILIC is illustrated as a fractionation technique. HILIC is a variant of normal phase liquid chromatography that overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. HILIC uses a hydrophilic stationary phase with a reversed phase eluent. Any polar chromatographic surface can be used for HILIC separations, including but not limited to simple unbonded silica, silanol, or diol bonded phases, amino or anion bonded phases, amide bonded phases, cation bonded phases, and zwitterion bonded phases. A typical mobile phase for HILIC chromatography includes acetonitrile (MeCN) with a small amount of water. However, any aprotic solvent miscible with water (e.g. THF) can be used. Alcohols can also be used, but their concentrations must be higher to achieve the same retention for the analytes compared to aprotic solvent-water combinations. Ionic additives such as ammonium acetate and ammonium formate are commonly used to control the pH and ionic strength of the mobile phase.

[0265] Samples can be prepared in a manner suitable for lipid extraction using methods known in the art. In one embodiment, a modified Bligh-Dyer protocol is used as described below. Add H2O to a constant volume / weight of sample in a 13x100mm glass screw-cap tube to a final volume of 1mL. Allow sample to stand at room temperature for 10 minutes. Add 2mL MeOH and 0.9mL CHCl2 to sample and vortex sample. If sample is not monophasic, add 50μl MeOH to sample and vortex (repeat as necessary until sample is monophasic). Add ULQS (i.e., internal standard), vortex, and incubate sample at room temperature for 30 minutes. Add 1mL H2O and 0.9mL CHCl2 to sample and mix by inversion. Centrifuge sample at 3400rpm for 15 minutes. Collect bottom layer from sample tube and transfer to new glass tube. Add 2 mL of CH2Cl2 to the sample remaining in the extraction tube and mix by inversion. Centrifuge the sample at 3400 rpm for 15 min. Collect the bottom layer and add to the first extraction solution. Evaporate the solvent (e.g., using a GeneVac) and resuspend the lipids in a 50:50 mixture of MeOH:CH2Cl2 (vol:vol) and use or store at -20°C until use. When ready to use, dilute the lipid sample with mobile phase A for LCMS analysis.

[0266] In this example, liquid chromatography (HILIC) is performed using a Sciex ExionLC with an AD pump with a Luna 3 μm NH2, 2×100 mm column (although any suitable instrument and column can be used). Mobile phases A and B are: Mobile phase A: 10:90 (vol:vol) CHCl3:ACN + 1 mM NH4AC; Mobile phase B: 50:50 (vol:vol) H2O:ACN + 1 mM NH4AC. Typical injection volume is 5 μl and column temperature is 35° C. In this example, a total runtime of 14.00 minutes is used with the following gradient:

[0267] [Table 9]

[0268] The liquid chromatography output is introduced directly into the mass spectrometer. In this example, MS is performed using a Sciex 6500+ Triple Quad mass spectrometer (although any suitable instrument and column may be used). The operating conditions are as follows:

[0269] [Table 10]

[0270] The MRM parameters were: MRM detection window--30 sec, and target scan time--0.3 sec.

[0271] Example 2 This example shows a representative distribution of ULQS internal standards for SM and PG lipids. This example shows HILIC classes showing concentration variations of internal standards that model the typical distribution of acyl chain length and saturation variations of naturally occurring lipids within the SM and PG classes. Figure 3 shows the differences in pattern abundance between the two ULQS SM and PG classes for different species within each class. Valley vs. peak shape. These spectra were obtained using HILIC based chromatography on a Sciex® Exion LC system with a Sciex® 6500+ triple quadrupole mass spectrometer.

[0272] In this example, the MS procedure used was as described in Example 1 using liquid chromatography (HILIC), except that mobile phase A was 10:90 (vol:vol) CHCl3:ACN + 2 mM NH4AC; mobile phase B: 50:50 (vol:vol) HO:ACN + 2 mM NH4AC.

[0273] Example 3 This example illustrates the advantage of having multiple internal standards for one or more classes of lipids. The presence of multiple ULQS internal standards accounts for the variation in acyl chain length and saturation / unsaturation of acyl chains observed in naturally occurring lipid species in biological systems. These differences are known to affect the ionization and fragmentation ability of lipids in a sample, and therefore generate different signal responses when using mass spectrometry techniques. The above often results in quantitative errors when only one internal standard is present in a sample and applied across multiple different natural species.

[0274] Figure 4 shows the calibration curves generated using five ULS QPS internal standards. Figure 4 shows the change in linearity for each ULQS PS internal standard species, demonstrating the need for multiple internal standards to account for the changes in acyl chain length and saturation / unsaturation observed in naturally occurring lipid species. The use of ULQS internal standards addresses the issue of variability observed in natural lipid species due to differences in ionization and fractionation fragmentation efficiencies. These concentration curves were obtained using HILIC based chromatography on a Sciex® Exion LC system with a Sciex® 6500+ triple quadrupole mass spectrometer.

[0275] In this example, the MS procedure used was as described in Example 1 using liquid chromatography (HILIC), except that mobile phase A was 10:90 (vol:vol) CHCl3:ACN + 2 mM NH4AC; mobile phase B: 50:50 (vol:vol) HO:ACN + 2 mM NH4AC.

Claims

1. A universal lipid quantification standard (ULQS) comprising a plurality of isotopically labeled lipid standards, the plurality of isotopically labeled lipid standards comprising: At least one lipid species from one or more lipid classes selected from the group consisting of lysophospholipid class, cholesterol ester class, triacylglycerol class, diacylglycerol class, ceramide class, and sphingomyelin class; and At least one lipid species from a phospholipid class comprising one or more phospholipid species selected from the group consisting of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), and phosphatidylinositol (PI). The above ULQS comprising:

2. Multiple isotope-labeled lipid standards were a. 15 phospholipid species, 10 lysophospholipid species, 3 cholesterol ester species, 5 triacylglycerol species, 3 diacylglycerol species, 3 ceramide species, and 3 sphingomyelin species; b. 20 phospholipid species, 15 lysophospholipid species, 5 cholesterol ester species, 9 triacylglycerol species, 5 diacylglycerol species, 5 ceramide species, and 5 sphingomyelin species; or c. 25 phospholipid species, 15 lysophospholipid species, 5 cholesterol ester species, 9 triacylglycerol species, 5 diacylglycerol species, 5 ceramide species, and 5 sphingomyelin species 2. The ULQS of claim 1 , comprising:

3. 3. The ULQS of claim 1 or 2, wherein the lipid species of each lipid class are selected to modify at least one of ionization efficiency, extraction efficiency, and differential fragmentation efficiency of the lipid species in the sample.

4. Multiple isotopically labeled lipid standards are provided having the general formula I: 【Chemistry 1】 [During the ceremony, R １ is -(CH ２ ) ｎ -N(CH ３ ) ３ , -(CH ２ ) ｎ -NH ３ , -(CH ２ ) ｎ -C(H)(NH ３ )-C(O)-O-,-(CH ２ ) ｎ -CH(OH)-CH(OH), inositol, and H; n is 1 to 6; R ２ is a C3-C30 saturated or unsaturated acyl chain; R ３ is a C3 to C30 saturated or unsaturated acyl chain; and R ４ is R ４ are independently an isotope of H or H, provided that at least one of is an isotope of H. The ULQS according to any one of claims 1 to 3, comprising at least one lipid species from at least one lipid species from a phospholipid class represented by:

5. 5. The ULQS of any one of claims 1 to 4, wherein the lysophospholipid class comprises one or more lysophospholipid species selected from the group consisting of lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylphosphatidylphosphoserine (LPS), lysophosphatidylglycerol (LPG), and lysophosphatidylinositol (LPI).

6. Multiple isotopically labeled lipid standards are represented by the general formula II: 【Chemistry 2】 [During the ceremony, R １ is -(CH ２ ) ｎ -N(CH ３ ) ３ , -(CH ２ ) ｎ -NH ３ , -(CH ２ ) ｎ -C(H)(NH ３ )-C(O)-O-,-(CH ２ ) ｎ -CH(OH)-CH(OH), inositol, and H; n is 1 to 6; R ２ is a C2-C24 saturated or unsaturated acyl chain; and R ４ is R ４ are independently an isotope of H or H, provided that at least one of is an isotope of H.

6. The ULQS of claim 5, comprising at least one lipid species from a lysophospholipid class comprising one or more lysophospholipid species represented by:

7. The plurality of isotopically labeled lipid standards comprises at least one lipid species selected from cholesterol ester species, wherein the cholesterol ester species has the general formula III: 【Chemistry 3】 [During the ceremony, R ２ is a C9 to C29 saturated or unsaturated acyl chain; and R ４ is R ４ are independently an isotope of H or H, provided that at least one of is an isotope of H. The ULQS according to any one of claims 1 to 6, which is represented by the formula:

8. The plurality of isotopically labeled lipid standards comprises at least one lipid species from a triacylglycerol class, where the triacylglycerol class comprises one or more triacylglycerol species, and where the triacylglycerol species has a general formula IV: 【Chemistry 4】 [During the ceremony, R ２ is a C3-C30 saturated or unsaturated acyl chain; R ３ are each independently a C3 to C25 saturated or unsaturated acyl chain; and R ４ is R ４ are independently an isotope of H or H, provided that at least one of is an isotope of H. The ULQS of claim 1, wherein the ULQS is represented by the formula:

9. The plurality of isotopically labeled lipid standards comprises at least one lipid species from a diacylglycerol class, where the diacylglycerol class comprises one or more diacylglycerol species, and where the diacylglycerol species has the general formula V: 【Chemistry 5】 [During the ceremony, R ２ is a C3-C30 saturated or unsaturated acyl chain; R ３ is a C3-C25 saturated or unsaturated acyl chain; and R ４ is R ４ are independently an isotope of H or H, provided that at least one of is an isotope of H. The ULQS according to any one of claims 1 to 8, which is represented by the following formula (I): or a pharma- ceutically acceptable salt thereof.

10. The plurality of isotopically labeled lipid standards comprises at least one lipid species from the ceramide class, where the ceramide class comprises one or more ceramide species, and the ceramide species has the general formula VI: 【Chemistry 6】 [During the ceremony, R ２ is a C10-C30 saturated or unsaturated acyl chain; R ４ is R ４ are independently an isotope of H or H, provided that at least one of is an isotope of H. The ULQS according to any one of claims 1 to 9, which is represented by the following formula (I): or a pharma- ceutically acceptable salt thereof.

11. The plurality of isotopically labeled lipid standards comprises at least one lipid species from the sphingomyelin class, where the sphingomyelin class comprises one or more sphingomyelin species, the sphingomyelin species having a general formula VII: 【Chemistry 7】 [During the ceremony, R １ teeth, 【Chemistry 8】 and R ２ is a C10-C30 saturated or unsaturated acyl chain; and R ４ and R ４ Each ' is independently R ４ Or R ４ is an isotope of H or H, provided that at least one of The ULQS according to any one of claims 1 to 10, which is represented by the following formula (I): or a pharma- ceutically acceptable salt thereof.

12. R １ but, 【Chemistry 9】 and R ４ The ULQS of claim 11, wherein

13. R １ but, 【Chemistry 10】 and at least one R ４ The ULQS of claim 12, wherein is an isotope of H.

14. 14. The ULQS of any one of claims 1 to 13, comprising at least one lipid species from each of the lysophospholipid class, cholesterol ester class, triacylglycerol class, diacylglycerol class, ceramide class, and sphingomyelin class.

15. 2. The ULQS of claim 1, comprising 5-40 phospholipid species, 5-25 lysophospholipid species, 3-15 cholesterol ester species, 5-20 triacylglycerol species, 3-15 diacylglycerol species, 3-15 ceramide species, and 3-15 sphingomyelin species.

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