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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.

Chlorinated phospholipids are formed by the reaction of hypochlorous acid (HOCl), generated by the enzyme myeloperoxidase under inflammatory conditions, and the unsaturated fatty acyl residues or the head group. In the first case the generated chlorohydrins are both proinflammatory and cytotoxic, thus having a significant impact on the structures of biomembranes. The latter case leads to chloramines, the properties of which are by far less well understood. Since HOCl is also widely used as a disinfecting and antibacterial agent in medicinal, industrial, and domestic applications, it may represent an additional source of danger in the case of abuse or mishandling. This review discusses the reaction behavior of in vivo generated HOCl and biomolecules like DNA, proteins, and carbohydrates but will focus on phospholipids. Not only the beneficial and pathological (toxic) effects of chlorinated lipids but also the importance of these chlorinated species is discussed. Some selected cleavage products of (chlorinated) phospholipids and plasmalogens such as lysophospholipids, (chlorinated) free fatty acids andα-chloro fatty aldehydes, which are all well known to massively contribute to inflammatory diseases associated with oxidative stress, will be also discussed. Finally, common analytical methods to study these compounds will be reviewed with focus on mass spectrometric techniques.

Background A rapid liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the quantification of reactive oxygen species (ROS) derived free oxysterols and cholesterol in human plasma and atherosclerotic plaque. Method In vitro autoxidation of cholesterol during sample pretreatment was avoided by applying only one protein precipitation and re-concentration step using 80 μl plasma. For preparation of 10 mg atherosclerotic plaques an additional liquid–liquid extraction was included. Free 7-keto-, 7-α/s-hydroxy-, 5,6-α-epoxy-, 5,6-β-epoxycholesterol, cholestane-3s,5α,6s-triol and cholesterol were separated within 7 min on a monolithic column. An API 4000 tandem mass spectrometer was applied in positive ionization mode using atmospheric pressure chemical ionization. Results The detection limit was 0.1 ng/ml and the linearity ranged from 0.5 to 0.75 to 2000 ng/ml for the oxysterols and from 50 to 1000 μg/ml for cholesterol. Recovery was between 80.9 and 107.9%. Between-run imprecision ranged from 7.9 to 11.7%. Analysis of plasma samples from additional 50 middle-aged volunteers revealed a large inter-individual variability (e.g. 7-ketocholesterol 2.63-30.47 ng/ml). Oxysterol concentrations normalized to cholesterol were about 43 times higher in carotid plaque compared to plasma (n = 5). Conclusion This rapid LC–MS/MS method enables reliable quantification focused on especially ROS-derived oxysterols in human plasma and atherosclerotic plaque samples under high-throughput conditions.