The analysis of lipids by mass spectrometry has been carried out for more than six decades. Fatty acids and simple derivatives such as methyl esters were some of the first lipids investigated in biochemical studies by electron ionization (EI) mass spectrometry. The use of electron ionization and the ancillary technique of gas chromatography for structural studies of simple and complex fatty acids have been extensively covered in a previous monograph (R. C. Murphy, Mass spectrometry of lipids, in Handbook of Lipid Research, ed. F. Snyder, Plenum Press, New York, 1993, vol. 7). The major limitations of EI and gas chromatography-mass spectrometry (GC-MS) have always been the requirement of volatility of the lipids in order to become ionized within the high vacuum of the ion source. This requirement is not an issue with electrospray ionization (or MALDI) since ionization occurs outside the high vacuum of the mass spectrometer and mass separation of ions is carried out by devices such as quadrupolal electrical fields, magnetic sectors, or time-of-flight sectors under high vacuum conditions. This feature of atmospheric ionization has an enormous advantage to the investigator, as made obvious by the explosive growth of electrospray ionization and the associated technique of liquid chromatography-mass spectrometry (LC-MS) that is now applied to a large number of very diverse lipid structures in biochemical studies. The combined liquid chromatography and mass spectrometer instrument is one established as being able to analyze virtually all types of lipid substances. Unlike the very energetic ionization techniques of electron ionization, electrospray ionization is quite gentle, leading to, in most cases, a single molecular ions species such as [M − H]⁻, [M + H]⁺ or [M + cation]⁺. The mechanism of electrospray ionization has been extensively studied and various reviews should be consulted to obtain a better understanding of this ionization process, since such details will not be covered in this monograph. MALDI ionization has also been applied to the analysis of virtually all lipids and the mechanism of ion formation extensively reviewed. Many of the general features of the behavior of electrospray generated molecular ion species in terms of mechanism of carbon–carbon bond cleavages directly apply to MALDI generated ions.

Both techniques generate fairly low energy ions that carry information of the intact molecular weight of the lipid. When a high resolution mass spectrometer, such as ion cyclotron resonance cell, orbitrap, the modern time-of-flight instrument are employed to measure the mass-to-charge ratio of the ion corresponding to the lipid molecular weight, one is able to calculate with excellent accuracy the elemental composition of the observed molecular ion species. However, structural details (unlike that often obtained by electron ionization) require subsequent excitation of the ion to a sufficiently high energy level to break covalent bonds. This is typically accomplished in a tandem mass spectrometer, where collision between a selected ion isolated by a mass separation device takes place with neutral gas molecules. Often several collisions are required for a covalent to bond break and resultant product ions are then analyzed by mass. It is the observed mass-to-charge ratios (m/z) and intensity of these product ions that carry attendant structural clues of the precursor ion.

The use of tandem mass spectrometry for the analysis of lipids requires experimental determination of the best instrumental conditions to employ to generate the ion(s) of interest. These conditions include voltages applied to ions for the optimal electrospray ion yield, collision gas and pressure in collision cell, collision energy (laboratory frame of reference), resolution of precursor ion section, and product ion selection to name a few conditions. All of these parameters need to be optimized and instrument companies provide essential guidelines. These topics will not be addressed in this book since they are typically empirically found. That is not to say they are not important, but this book deals with the chemistry of product ion formation. The product ions discussed here are ones that should be observed with any instrument. However, the exact ratio of ions will likely differ between instrument and analysis conditions. The most variable ion abundance is that of the precursor mass or mass selected for collisional activation. One can collisionally activate all precursor ions so that no ion at this mass-to-charge ratio appears in the product ion spectrum. I have chosen to retain a fair amount of precursor ions in the figures generated for this book. This required in some cases to reduce the collision energy for certain lipids. References are provided for all lipids discussed in this monograph and the primary literature should be consulted to gain insight into the optimal mass spectrometer parameters to employ.

In the early 1990s, I completed the monograph Mass Spectrometry of Lipids, which covered, in detail, various mass spectrometric approaches to analyze complex lipids from fatty acids to phospholipids. For the vast majority of examples of lipid analysis presented, electron ionization and the closely related ionization technique chemical ionization were presented. Complex lipids that could not pass underivatized through a gas chromatograph were analyzed at that time by fast atom bombardment ionization. Electrospray ionization and MALDI were just in the early phases of development, but the promise was clearly there.

In the intervening two decades we have seen a revolution in biochemistry driven largely by electrospray ionization of proteins and peptides (proteomics) using LC-MS/MS instrumentation. The impact on lipid biochemistry has been equally revolutionary. This monograph will cover this ionization technique applied to the large number of classes of lipids that exist in nature due to enzymatic catalyzed processes. At the heart of understanding electrospray ionization and application to problems in biochemistry, I feel one must come to grips with events taking place with ions while in the mass spectrometer sectors. In other words, to begin to understand the gas phase ion chemistry taking place, not only following collisional activation (collision induced dissociation, CID), but also during the ionization process itself. Therefore, with each lipid class example, I will try to suggest mechanisms by which the observed product ions are formed if mechanisms have not been examined in detail in previous publications. In most cases, these events follow fairly simple chemical rules and it is hoped that by working with specific examples, the reader can expand their facility of understanding ions derived from complex lipids and then apply these principles to more complex lipids and to solve complex biochemical problems. In some cases, these suggestions as to mechanism will be in error but it is hoped that my suggestions will encourage investigators to examine, in detail, the protons being rearranged and bonds broken and formed, then to devise experiments using stable isotope labeling, theoretical calculations, and chemical synthesis of intermediates, so that the correct mechanism can be revealed.

Robert C. Murphy,

University of Colorado, Aurora, CO, USA