Direct Infusion MS (ESI)

Low mass resolution

Identification of lipid species with low mass resolution instruments requires tandem mass spectrometry (typically triple quadrupole instruments), utilizing typical fragments and MRM transitions  for lipid classes and molecular species.

Glycerophospholipids
  • Annotation of lipid class (= lipid species level or sum composition) requires:
    • Detection of lipid class specific fragment
    • Assumptions applied for annotation have to be shown
    • Examples:
      • PIS+ m/z 184: Annotation of signal at m/z 746.6 as PC O-34:1; based on the assumption that only acyl chains with even number of C-atoms
      • PIS+ m/z 184: Annotation of signal at m/z 746.6 as PC 33:1; based on the assumption that only ester bonds are present
      • PIS+ m/z 196: Annotation of signal at m/z 702.5 as PE O-34:1; based on the assumption that only acyl chains with even number of C-atoms
      • PIS+ m/z 196: Annotation of signal at m/z 702.5 as PE 33:1; based on the assumption that only ester bonds are present
  • Annotation of acyl/alkyl chains (= fatty acyl level or molecular lipid species e.g. PC 16:0_18:1) requires:
    • Detection of lipid molecular species specific fragments
    • Diacyl: detection of fragments for both acyl chains and/or NL of the acyl chains (as fatty acid or ketene)
    • Akyl-acyl: detection of fragments for only one acyl chain and/or NL of this acyl chains (as fatty acid or ketene)
    • Examples:
      • Product ions (-) of m/z 804.6 – m/z 255.2 and 281.2 – annotation as PC 16:0_18:1  [M+HCOO]–
      • Product ions (-) of m/z 716.5 – m/z 255.2 and 281.2 – annotation as PE 16:0_18:1  [M-H]–
      • Product ions (-) of m/z 760.6 – m/z 478.3 and 504.3 and/or m/z 496.3 and 522.3 annotation as PC 16:0_18:1 [M+H]+
  • Annotation of lysophospholipids (= fatty acyl level or molecular lipid species) requires:
    • Detection of lipid class specific fragment or lipid molecular species specific fragment
    • Assumptions applied for annotation have to be shown
    • Examples:
      • PIS+ m/z 184: Annotation of signal at m/z 482.6 as LPC O-16:0; based on the assumption that only acyl chains with even number of C-atoms
      • PIS+ m/z 184: Annotation of signal at m/z 482.6 as LPC 15:0; based on the assumption that only ester bonds are present
Sphingolipids
  • Annotation of lipid class (= lipid species level or sum composition) requires:
    • Detection of lipid classes specific fragment
    • Assumptions applied for annotation have to be shown
    • Examples:
      • PIS+ m/z 184: Annotation of signal at m/z 703.6 as SM 34:1; based on the assumption of sphingoid base with two hydroxyl-groups
      • PIS+ m/z 184: Annotation of signal at m/z 703.6 as SM 33:2; based on the assumption of sphingoid base with three hydroxyl-groups
  • Annotation of acyl chains/sphingoid bases (= fatty acyl level or molecular lipid species) requires:
    • Detection of lipid molecular species specific fragment
    • Examples:
      • PIS+ m/z 266: Annotation of signal at m/z 540.5 as Cer d18:0/16:0
      • PIS+ m/z 264: Annotation of signal at m/z 538.5 as Cer d18:1/16:0
      • PIS+ m/z 264: Annotation of signal at m/z 700.6 as HexCer d18:1/16:0
      • PIS+ m/z 264: Annotation of signal at m/z 862.6 as Hex2Cer d18:1/16:0
      • PIS+ m/z 262: Annotation of signal at m/z 536.5 as Cer d18:2/16:0
Sterols
  • Annotation of lipid class (= lipid species level or sum composition) requires:
    • Detection of lipid classes specific fragment
    • Assumptions applied for annotation have to be shown
    • Examples:
      • PIS+ m/z 369: Annotation of signal at m/z 666.6 as CE 18:2 [M+NH4]+; based on the assumption that only ester bonds are present and that cholesterol is the major sterol St 27:1_OH

High mass resolution

  • Identification power depends on mass resolution and separation of isobaric ions
  • Identification based on accurate mass only is only sufficient:
    • in combination with a thorough data deconvolution
    • when additional evidence from both ion modes or other methods like MS/MS justifies the findings
  • Identification (at sufficient mass resolution) allows differentiation of bond types (acyl/alkyl) number of hydroxyl groups
  • Identification of acyl/alkyl chains requires MS/MS and detection of respective fragment ions as described for low mass resolution
  • Examples:
    • HR-MS+: Annotation of m/z 760.5850 as PC 34:1 when PE 37:1 could be excluded based on lipid biochemistry and/or m/z 804.5760 is detected in negative ion mode with formate containing solvent
    • HR-MS+: Annotation of m/z 746.6058 as PC O-34:1 when PE O-37:1 could be excluded based on lipid biochemistry and/or m/z 790.5967 is detected in negative ion mode with formate containing solvent