Analytical and Bioanalytical Chemistry

, Volume 410, Issue 9, pp 2437–2447 | Cite as

The combination of 2,5-dihydroxybenzoic acid and 2,5-dihydroxyacetophenone matrices for unequivocal assignment of phosphatidylethanolamine species in complex mixtures

  • Jenny Schröter
  • Annabelle Fülöp
  • Carsten Hopf
  • Jürgen Schiller
Research Paper


Unequivocal assignment of phospholipid peaks in complex mixtures is difficult if only the m/z values but no tandem mass spectrometry (MS/MS) data are available. This is usually the case for matrix-assisted laser/desorption ionization time-of-flight (MALDI-TOF) MS imaging experiments and the analysis has normally to be performed without prior separation. Another problem might be the often matrix-induced loss of one methyl group in phosphatidylcholine (PC) species, which makes them detectable as negative ions becoming isomers of some phosphatidylethanolamines (PEs). Selected lipid mixtures of known compositions were investigated by negative ion MALDI-TOF MS and various imaging experiments. In addition to common matrices such as 2,5-dihydroxybenzoic acid (DHB) and 9-aminoacridine (9-AA), different binary matrices, including 2,5-dihydroxyacetophenone (2,5-DHAP) as matrix additive to DHB, were tested to probe their performance in both ionization modes. Beside artificial PC and PE mixtures of known compositions, egg yolk and liver extracts as well as cryosections from liver and pancreas tissue were selected as biologically relevant systems. The majority of the binary MALDI matrices used here leads to the loss of a methyl group from PC in the negative ion mode, which makes the clear identification of PE species ambiguous. However, this problem does not apply if a mixture of DHB and 2,5-DHAP is used. Therefore, the application of DHB/2,5-DHAP as matrix is a simple method to unequivocally identify PEs even in complex mixtures and tissue sections as negative ions and without the necessity to separate the individual lipid classes prior to MS detection.

Graphical abstract

Many common MALDI matrices (such as 9-AA) induce the loss of a methyl group from PC rendering the PC detectable as negative ion. These ions (m/z 744.6 in the upper trace) represent isomers of typical PE species. It will be shown that this problem can be avoided if mixtures between DHB and 2,5-DHAP are applied. At these conditions, POPC is exclusively detectable as a matrix adduct with DHB (at m/z 912.6, lower trace) and does not interfere with PE. This approach can also be used in MALDI MS imaging.


MALDI-TOF MS MALDI matrix MALDI imaging Phospholipids NMR 2,5-Dihydroxyacetophenone 



This study was supported by the German Research Council (DFG Schi 476/12-2, DFG Schi 476/16-1, and SFB 1052/Z3). We would also like to thank all our colleagues who helped us perform related experiments. We are particularly indebted to Bruker Daltonik GmbH (Bremen, Germany) and Merck Millipore (Darmstadt, Germany) for the continuous and generous support.

Funding information

C.H. thanks the Deutsche Forschungsgemeinschaft (DFG) for the funding of a SolariX FTICR MS (INST 874/7-1 FUGG).

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Supplementary material

216_2018_926_MOESM1_ESM.pdf (1.3 mb)
ESM 1 (PDF 1292 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Faculty of Medicine, Institute for Medical Physics and BiophysicsLeipzig UniversityLeipzigGermany
  2. 2.Center for Biomedical Mass Spectrometry and Optical Spectroscopy (CeMOS)Mannheim University of Applied SciencesMannheimGermany

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