Complementary approach for analysis of phospholipids by liquid chromatography hyphenated to elemental and molecular mass spectrometry

Phospholipids are one of the most important lipid categories with multiple functions in biological systems. Their analysis can contribute to a better understanding of metabolomic and kinetic processes in living cells. Comprehensive methods based on liquid chromatography coupled to mass spectrometry are available for phospholipid identification and quantification. However, quantification of phospholipids using electrospray ionization‐mass spectrometry with internal standards is still challenging due to several reasons. In particular, the detector response of phospholipid species differs with variation of the head group as well as the fatty acid chain length and double bond number. Inductively coupled plasma‐tandem mass spectrometry (ICP‐MS/MS) provides an alternative approach for their absolute quantification with universal detector response for phosphorus independent of its chemical form and proportional to its quantity. Therefore, a quantification method based on compound‐independent calibration using hydrophilic interaction liquid chromatography (HILIC) coupled to ICP‐MS/MS was developed. An inverse gradient system was implemented for constant mobile phase composition after HILIC separation, which provides steady plasma ionization conditions. Isobaric phosphorus interferences were decreased by using the oxygen reaction mode of the triple quadrupole based ICP‐MS/MS instrument. Complementary molecular information was obtained by ESI‐high‐resolution MS and MS/MS. The applicability of this approach was demonstrated in a proof of concept by complementary analysis of a total lipid extract of baker's yeast.