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EP-3852896-B1 - SYSTEM AND METHOD FOR LIPID QUANTIFICATION

EP3852896B1EP 3852896 B1EP3852896 B1EP 3852896B1EP-3852896-B1

Inventors

  • GETHINGS, LEE
  • ISAAC, Giorgis, M.
  • MUNJOMA, Nyasha, Clarence
  • PLUMB, ROBERT, S.
  • RAINVILLE, PAUL

Dates

Publication Date
20260506
Application Date
20190919

Claims (12)

  1. A method for identifying and quantifying lipids in a biological sample comprising classes of lipids, the method comprising: providing a library that comprises multiple reaction monitoring (MRM) transitions for individual lipids within the classes of lipids in the biological sample; combining known concentrations of stable isotope labeled (SIL) lipid standards with the biological sample to form a sample matrix, wherein the sample matrix includes at least one SIL lipid standard for each class of lipid in the biological sample that is being detected; introducing the sample matrix into a liquid chromatography (LC) system to separate lipids in the sample matrix into discrete bands of lipids, wherein each discrete band of lipids comprises lipids of the same class, and wherein each class of lipids appears in a single band; directing each of the separated discrete classes of lipids to a mass spectrometry (MS) system configured to obtain MRM transitions of the separated lipids ; identifying lipids in the sample matrix by comparing the obtained MRM transitions to MRM transitions in the library; and quantifying the identified lipids in each discrete class using the known concentrations of the SIL lipid standards characterised in that : the biological sample comprises lysophosphatidylcholines (LPC), phosphatidylcholines (PC), and sphingomyelins (SM); the SIL lipid standards comprise a LPC, a PC, and a SM; and wherein the LC system separates the lipids in the sample matrix into three discrete bands, the first discrete band comprising PCs, the second discrete band comprising SMs and the third discrete band comprising SM.
  2. A method for identifying and quantifying lipids in a biological sample comprising classes of lipids, the method comprising: providing a library that comprises multiple reaction monitoring (MRM) transitions for individual lipids within the classes of lipids in the biological sample; combining known concentrations of stable isotope labeled (SIL) lipid standards with the biological sample to form a sample matrix, wherein the sample matrix includes at least one SIL lipid standard for each class of lipid in the biological sample that is being detected; introducing the sample matrix into a liquid chromatography (LC) system to separate lipids in the sample matrix into discrete bands of lipids, wherein each discrete band of lipids comprises lipids of the same class, and wherein each class of lipids appears in a single band; directing each of the separated discrete classes of lipids to a mass spectrometry (MS) system configured to obtain MRM transitions of the separated lipids ; identifying lipids in the sample matrix by comparing the obtained MRM transitions to MRM transitions in the library; and quantifying the identified lipids in each discrete class using the known concentrations of the SIL lipid standards characterised in that : the biological sample comprises triradylglycerolipid (TG), ceramides (CER), hexosylceramides (HexCer) and sphingomyelins (SM); the SIL lipid standards comprise a triradylglycerolipid (TG) and a SM; and wherein the LC system separates the lipids in the sample matrix into two discrete bands, the first discrete band comprising TGs and the second discrete band comprising CERs, HexCers, and SMs.
  3. A method for identifying and quantifying lipids in a biological sample comprising classes of lipids, the method comprising: providing a library that comprises multiple reaction monitoring (MRM) transitions for individual lipids within the classes of lipids in the biological sample; combining known concentrations of stable isotope labeled (SIL) lipid standards with the biological sample to form a sample matrix, wherein the sample matrix includes at least one SIL lipid standard for each class of lipid in the biological sample that is being detected; introducing the sample matrix into a liquid chromatography (LC) system to separate lipids in the sample matrix into discrete bands of lipids, wherein each discrete band of lipids comprises lipids of the same class, and wherein each class of lipids appears in a single band; directing each of the separated discrete classes of lipids to a mass spectrometry (MS) system configured to obtain MRM transitions of the separated lipids ; identifying lipids in the sample matrix by comparing the obtained MRM transitions to MRM transitions in the library; and quantifying the identified lipids in each discrete class using the known concentrations of the SIL lipid standards characterised in that : the biological sample comprises monoradylglycerolipids (MG), diradylglycerolipids (DG), triradylglycerolipids (TG), lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE), phosphatidylcholines (PC), sphingomyelins (SM), phosphatidic acids (PA), phosphatidylethanolamines (PE), phosphatidylglycerols (PG), phosphatidylinositols (PI), cholesterol, and cholesterol ester; the SIL lipid standards comprise a MG, a DG, a TG, a LPC, a LPE, a PC, a SM, a PA, a PE, a PG, a PI, cholesterol, and a CE; and wherein the LC system separates the lipids in the sample matrix into eight discrete bands: the first discrete band comprising MGs, DGs, TGs, cholesterol and cholesterol ester; the second discrete band comprising PGs; the third discrete band comprising PCs; the fourth discrete band comprising PEs; the fifth discrete band comprising SMs; the sixth discrete band comprising LPCs; the seventh discrete band comprising LPEs, and the eighth discrete band comprising PIs and PAs.
  4. A method for identifying and quantifying lipids in a biological sample comprising classes of lipids, the method comprising: providing a library that comprises multiple reaction monitoring (MRM) transitions for individual lipids within the classes of lipids in the biological sample; combining known concentrations of stable isotope labeled (SIL) lipid standards with the biological sample to form a sample matrix, wherein the sample matrix includes at least one SIL lipid standard for each class of lipid in the biological sample that is being detected; introducing the sample matrix into a liquid chromatography (LC) system to separate lipids in the sample matrix into discrete bands of lipids, wherein each discrete band of lipids comprises lipids of the same class, and wherein each class of lipids appears in a single band; directing each of the separated discrete classes of lipids to a mass spectrometry (MS) system configured to obtain MRM transitions of the separated lipids ; identifying lipids in the sample matrix by comparing the obtained MRM transitions to MRM transitions in the library; and quantifying the identified lipids in each discrete class using the known concentrations of the SIL lipid standards characterised in that : the biological sample comprises lysophosphatidylethanolamines (LPE), phosphatidylethanolamines (PE), phosphatidylglycerols (PG), and phosphatidylinositols (PI); the SIL lipid standards comprise a LPE, a PE, a PG, and a PI; and wherein the LC system separates the lipids in the sample matrix into four discrete bands, the first discrete band comprising PGs, the second discrete band comprising PEs, the third discrete band comprising LPEs and the fourth discrete band comprising PIs.
  5. The method of any one of claims 1 to 4, wherein the LC system is a hydrophilic interaction chromatography (HILIC) system.
  6. The method of any one of claims 1 to 4, wherein the MRM transitions are performed in both positive and negative ion mode.
  7. The method of claim 6, wherein the MRM transitions can be fatty acyl chain fragments, head group fragments, or neutral loss fragments.
  8. The method of any one of claims 1 to 4, further comprising: extracting the lipids from the sample matrix includes using protein precipitation with pre-cooled isopropanol.
  9. The method of any one of claims 1 to 4, wherein the library further comprises liquid chromatography (LC) conditions for separating classes of lipids and retention times for each class of lipids present in the sample matrix, and wherein the classes of lipids are separated using the LC conditions obtained from the library.
  10. The method of any one of claims 1 to 4, wherein the library further comprises mass spectrometry (MS) conditions for obtaining MRM transitions for each class of lipids, and wherein the MRM transitions for each class of lipids are generated using the MS conditions obtained from the library.
  11. The method of any one of claims 1 to 4 for identifying potential biomarkers, the method comprising: obtaining a number of biological samples associated with a medical condition, the biological samples comprising different classes of lipids; and determining a relationship between the identified and quantified lipids and the medical condition.
  12. The method of any one of claims 1 to 4 for diagnostic screening for a medical condition associated with one or more lipids, the method comprising: obtaining a biological sample associated with the medical condition, the biological sample comprising different classes of lipids; and identifying the medical condition based on the identified and quantified lipids in the biological sample.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to United States Provisional Patent Application No. 62/734,410 filed on September 21, 2018 titled "SYSTEM AND METHOD FOR LIPID QUANTIFICATION". FIELD OF THE TECHNOLOGY The present disclosure generally relates to systems and methods for identifying and quantifying lipids. In particular, the present disclosure relates to systems and methods for identifying and quantifying lipids using a chromatography system coupled to a mass spectrometer. BACKGROUND Chromatography involves the flowing of a mobile phase over a stationary phase to effect separation and is often combined with a detector, such as a mass spectrometer. Advances in MS have allowed for more in-depth analysis of lipids; however, unambiguous identification and quantification has proven difficult, as lipids exhibit a high number of isomeric and isobaric lipid species. Furthermore, MS spectra often contain peaks and fragments from multiple compounds, making confident identification and relative quantitation of specific molecular species difficult and time consuming. As a result, the transfer of lipidomic data between laboratories is severely hindered, making it problematic to draw biological interpretation with multiple-site studies. WO2017/210097 discloses a mass spectrometry method for detection and quantitation of metabolites , US2017/160264 discloses the use of ceramides and lpls in diagnosing cvd. M. Scherer et al, "A rapid and quantitative LC-MS/MS method to profile sphingolipids", J. Lipid Res. 51(7), pages 2001-2011 (2010) discloses a method for simultaneous quantification of sphingolipid classes. Appropriate internal standards were added prior to lipid extraction. SUMMARY Identification and quantification of lipids raises a number of challenges due to the high number of isomeric and isobaric lipid species. Technology for unambiguously and efficiently identifying and quantifying lipids would be beneficial and highly desirable. The present invention provides a method for identifying and quantifying lipids in a biological sample comprising classes of lipids according to any one of claims 1 to 4, wherein any of these methods are used for identifying potential biomarkers according to claim 11, and for diagnostic screening for a medical condition associated with one or more lipids according to claim 12. In one aspect, the present technology relates to a method for screening lipids. The method includes selecting a set of standards to identify at least one class of lipids; combining the standards with a biological sample to form a sample matrix; introducing the sample matrix into a chromatography system to separate the at least one class of lipids; directing the separated lipids to a detector; generating a calibration curve with known concentration of the set of standards; plotting a detector response of the separated lipids against the known concentrations of the set of standards; and quantifying the separated lipids based on a comparison between the detector response and the calibration curve. The detector is configured to perform targeted quantification of the separated lipids using multiple reaction monitoring (MRM) transitions. In another example embodiment, the MRM transitions are performed in both positive and negative ion mode. In another example embodiment, the MRM transitions can be fatty acyl chain fragments, head group fragments, or neutral loss fragments. In another example embodiment, the class of lipids includes monoradylglycerolipids (MG), diradylglycerolipids (DG), triradylglycerolipids (TG), ceramides, lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE), phosphatidylcholines (PC), sphingomyelins (SM), free fatty acids (FFA), lysophosphatidylinositols (LPI), phosphatidic acids (PA), lysophosphatidic acids (LPA), phosphatidylethanolamines (PE), phosphatidylglycerols (PG), phosphatidylinositols (PI), phosphatidylserines (PS), cholesterol, cholesterol ester, hexsocyl ceramides, dihexsocyl ceramides, lipoprotein(a) (LPA), lypopolysaccharides (LPS), or lysyl-phosphatidylglycerol (LPG). In another example embodiment, the chromatography system is a hydrophilic interaction chromatography (HILIC) system. In another aspect, the present technology relates to a method for identifying potential biomarkers. The method includes selecting a set of standards to identify at least one class of lipids; combining the set of standards with a number of biological samples associated with a medical condition to form a sample matrix; introducing the sample matrix into a chromatography system to separate the at least one class of lipids; directing the separated lipids to a detector; generating a calibration curve with known concentrations of the set of standards; plotting a detector response of the separated lipids against the known concentrations of the set of standards; quantifying the separated lipids based on a comparison between the detector response