Matrix lipid analysis

Perres, N., Sajdera, S. W., Anderson, N. G Lipid analysis of vesicles isolated from the matrix of calcifying cartilage. Fed. Proc. 30, 1244 (1971)... 

The dynamic development of mass spectrometry has had a huge impact on lipid analysis. Currently, a variety of suitable mass spectrometers is available. In principal, a mass spectrometer consists of an ion source, a mass analyzer, and an ion detector. The typical features of each instrument (Fig. 2) result mostly from the types of ion source and mass analyzer. To date, the ionization techniques apphed to lipid analysis include Electrospray Ionization (ESI or nano-ESI), Atmospheric Pressure Chemical Ionization (APCI), Matrix-Assisted Laser Desorption/Ionization... 

Schiiier, J., Amhoid, J., Benard, S., Muller, M., Reichl, S. and Arnold, K., Lipid analysis by matrix-assisted laser desorption and ionization mass spectrometry A methodological approach. Anal Biochem, 267 (1999) 46-56. 

As noted above, PC is the by far most frequently investigated phospholipid [40, 82, 83], and consequently was one of the first PLs to be studied using MALDI-TOF-MS [46, 75]. As the pioneer in this field, Harvey [46] investigated, as early as 1995, a variety of PLs to determine which matrix would provide the best spectral quality. Among other matrices, a-cyano-4-hydroxycinnamic acid, 6,7-dihydroxy-coumarin and DH B gave the best results. Today, DHB is the most frequently used matrix for lipid analysis by MALDI-MS, and is regarded as the work horse of the field because essentially all Hpids-irrespective of their structure-are detectable with DH B. A comprehensive list of usefijl matrices for the lipid field is available in Refs [41] and [84]. 

For the detailed analysis of complex mixtures, a previous separation of the sample into the individual lipid classes is required, however, in the same manner as if a LC/MS process was being used. For this purpose, combined TLC/MALDI may be employed because, when the sample is dispersed in a solid matrix (i.e., the silica gel), it can easily be combined with a MALDl ion source. Thus, MALDI-M S can be expected to become a true alternative to currently established methods of lipid analysis. 

DESI offers numerous advantages for lipid analysis including little or no sample preparation, not requiring the addition of a matrix, atmospheric pressure ionization outside the mass spectrometer, and readily ionizing many lipid species. Thus, this technology can be conveniently used for the direct analysis and imaging of those ionizable lipid species present in biological samples such as tissue [105, 107-109]. This topic is extensively described in Chapter 12. 

Matrix compound(s) are usually ionized, which result in complications for lipid analysis in the low m/z region, such as the fragments that result from postsource decay, and are used for characterization of lipid structures. 

Another advantage of IMS is that endogenous lipid analysis directly from tissue sections can be performed with minimal sample preparation, particularly in the case of IMS by DESI. Usually, sample preparation for IMS only requires a short tissue wash followed by matrix application (e.g., for MALDI and SIMS). 

Angel, P.M., Spraggins, J.M., Baldwin, H.S. and Caprioli, R. (2012) Enhanced sensitivity for high spatial resolution lipid analysis by negative ion mode matrix assisted laser desorption ionization imaging mass spectrometry. Anal. Chem. 84, 1557-1564. 

Although several technologies have been used in lipidomics to identify, quantify, and understand the structure and function of lipids in biological systems, it is clear that the progress of lipidomics has been accelerated by the development of modern mass spectrometry (e.g., electrospray ionization (ESI) and matrix-assisted laser desorption/ionization). Mass spectrometric analysis of lipids plays a key role in the discipline. Therefore, this book is focused on the mass spectrometry of lipids that has occurred in these years. Other technologies for analysis of lipids, particularly those with chromatography, can be found in the book entitled Lipid Analysis Isolation, Separation, Identification and Lipidomic Analysis written by Drs William W. Christie and Xianlin Han. Readers who are interested in classical techniques and applications of mass spectrometry for analysis of lipids should refer to Dr Robert C. Murphy s book entitled Mass Spectrometry of Lipids. 

Among other techniques, reversed-phase TLC has been successfully applied to the separation of triacylglycerols, while silver-ion TLC has become one of the standard methods for the determination of cocoa butter equivalents in confectionery fats TLC has been used successfully in conjunction with matrix-assisted laser desorption—ionization mass spectrometry, as well. Theory and applications of TLC in lipid analysis have been reviewed by Christie and Han and others in books and reviews, to which the reader is referred [11,36—38]. 

Triple quadrupole MS instruments have been the most common ones in studies involving lipid analysis, butnovel hybrid (quadrupoletime-of-flight, etc.)instruments are rapidly gaining popularity due to their ability for multiple precursor ion scans simultaneously. Besides ESI, atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), and matrix-assisted laser desorption ionization (MALDI) have been employed in analysis of lipids. However, these methods seem to have an advantage over ESI only in special cases. For instance, APPI and APCI allow analysis of sterols without derivatization, which is needed for ESI. 

Gel permeation ehromatography (GPC)/normal-phase HPLC was used by Brown-Thomas et al. (35) to determine fat-soluble vitamins in standard referenee material (SRM) samples of a fortified eoeonut oil (SRM 1563) and a eod liver oil (SRM 1588). The on-line GPC/normal-phase proeedure eliminated the long and laborious extraetion proeedure of isolating vitamins from the oil matrix. In faet, the GPC step permits the elimination of the lipid materials prior to the HPLC analysis. The HPLC eolumns used for the vitamin determinations were a 10 p.m polystyrene/divinylbenzene gel eolumn and a semipreparative aminoeyano eolumn, with hexane, methylene ehloride and methyl tert-butyl ether being employed as solvent. 

As with urine, saliva (spumm) is easy to collect. The levels of protein and lipids in saliva or spumm are low (compared to blood samples). These matrices are viscous, which is why extraction efficiency of xenobioties amoimts to only 5 to 9%. By acidifying the samples, extraction efficiencies are improved as the samples are clarified, and proteinaceous material and cellular debris are precipitated and removed. Some xenobioties and their metabohtes are expressed in hair. Hair is an ideal matrix for extraction of analytes to nonpolar phases, especially when the parent xenobioties are extensively metabolized and often nondetectable in other tissues (parent molecules of xenobioties are usually less polar than metabolites). Hair is a popular target for forensic purposes and to monitor drug compliance and abuse. Human milk may be an indicator of exposure of a newborn to compounds to which the mother has been previously exposed. The main components of human milk are water (88%), proteins (3%), lipids (3%), and carbohydrates in the form of lactose (6%). At present, increasing attention is devoted to the determination of xenobioties in breath. This matrix, however, contains only volatile substances, whose analysis is not related to PLC applications. 

Although ribosomal proteins are readily observed as in Figures 13.7 and 13.8 altered matrix conditions can alter the relative ionization of bacterial whole-cell compounds. A systematic analysis involving laser power/fluence and sample preparation conditions reveals that if the concentrated trifluo-roacetic acid is added and the laser power increased above optimal conditions, ionization of bacterial surface compounds can be enhanced. Figure 13.9 is the resulting 9.4 T MALDI-FTMS, seen are both the Braun s lipoprotein56,57 and the Murein lipoprotein. Both of these compounds are complex combinations of hydrocarbon lipids attached to a protein base. This is the first MALDI-FTMS observation of surface proteins desorbed directly from whole cells by influencing ionization conditions. 

The potential for the preservation of lipids is relatively high since by definition they are hydrophobic and not susceptible to hydrolysis by water, unlike most amino acids and DNA. A wide range of fatty acids, sterols, acylglycerols, and wax esters have been identified in visible surface debris on pottery fragments or as residues absorbed into the permeable ceramic matrix. Isolation of lipids from these matrices is achieved by solvent extraction of powdered samples and analysis is often by the powerful and sensitive technique of combined gas chromatography-mass spectrometry (GC-MS see Section 8.4). This approach has been successfully used for the identification of ancient lipid residues, contributing to the study of artifact... 

A representative gas chromatogram with ECD of the analysis of various polar chlorinated pesticides isolated from cod liver oil [179] is shown in Fig. 13. Determination of the polar chlorinated pesticides in cod liver oil required clean up of the lipid matrix with a dimethylformamide/water/hexane liquid-liquid partitioning procedure followed by isolation using a normal-phase LC procedures, and final analysis by GC-ECD [179]. 

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