Lipid, analysis detection

The application of high-sensitivity ICP-MS detectors coupled to HPLC has enabled the detection of trace arsenic compounds present in marine animals. Thus, arsenocholine has been reported as a trace constituent (<0.1% of the total arsenic) in fish, molluscs, and crustaceans (37) and was found to be present in appreciable quantities (up to 15%) in some tissues of a marine turtle (110). Earlier reports (46,47) of appreciable concentrations of arsenocholine in some marine animals appear to have been in error (32). Phosphatidylarsenocholine 45 was identified as a trace constituent of lobster digestive gland following hydrolysis of the lipids and detection of GPAC in the hydrolysate by HPLC/ICP-MS analysis (70). It might result from the substitution of choline with arsenocholine in enzyme systems for the biogenesis of phosphatidylcholine (111). 

Caboni, M.F. and Rodriguez-Estrada, M.T. 1997. High-performance liquid chromatography coupled to evaporative light scattering detection in lipid analysis Some application. Seminars in Food Analysis 2 159-169. 

Low-wavelength UV detection (200-210 nm) is more sensitive and permits the use of gradients but precludes the use of certain common lipid solvents, such as chloroform and acetone, which are opaque in the UV region of interest. With low-wavelength UV detection, the response will also be somewhat dependent on fatty acid composition. For these reasons the mobile phases used in lipid analysis by HPLC may seem rather strange to workers familiar with the Thin Layer Chromatography (TLC) or open column separations. 

First, almost all studies of insect cuticular lipids have used gas chromatography (GC) to analyze lipid extracts, using standard GC conditions that only allow compounds under C40 to be detected. More specialized GC equipment that can extend this range to >C60, in particular, columns that can withstand high temperatures (>400°C) are now available. However, they have not yet found routine use for cuticular lipid analysis, despite recent studies that have clearly demonstrated that cuticular lipids do indeed contain hydrocarbons, waxes, and other compounds with molecular weights above 500 daltons (Cvacka et ai, 2006). Thus, to date, many studies may only have examined subsets of the cuticular components. 

Today, HPLC is the dominant analytical technique used for the analysis of most classes of compounds. The analyses can be carried out at room temperature and the collection of fractions for reanalysis or further manipulation is straightforward. The main reason for the slow acceptance of the HPLC technique for Upid analysis has been the detection system. Traditionally, HPLC used ultraviolet/visible (UV/vis) detection, which requires the presence of a chromophore in the analyte. Most lipid molecules do not contain chromo-phores and therefore would not be detected by UV/vis. Modern HPLC detection techniques, such as the use of a mass spectrometer as the detector, derivatization techniques to introduce chromophores, and the availability of pure solvents to reduce interference, have allowed HPLC to compete with and/or complement GC and other traditional methods of lipid analysis. In addition to analytical HPLC, preparative HPLC has been used extensively to collect pure samples of the lipids for the derivatization or synthesis of new compounds. 

Finally, the detection by liquid chromatograph-mass spectrometer (LC-MS), which has been largely dependent on the price, mode of ionization, and ease of operation of the mass spectrometer, is becoming popular [8]. It is predicted that LC-MS will become the method of choice for lipid analysis in coming years. 

Samples of lipids may be derivatized either before they are injected into the HPLC or after they emerge from the HPLC column. The purpose of derivatization is either to make the lipid molecules detectable or to improve peak shape during the analysis. The addition of functional groups, such as phenyl, 2,4-dinitrophenyl, or... 

In the case of cationic lipid/DNA complexes, the lipid markers that have been used are often the same as those used for conventional liposomes (i.e., CHE). This may not be appropriate. Therefore, we believe it is best to use a radiolabeled form of the lipids used to prepare the carrier system (133). Ideally, carrier cationic lipid and plasmid DNA quantification should be performed on the same tissues within in the same experiment. It may also be useful to analyze the fate of the neutral lipid components of the carrier. Radiolabeled DOPE is available, for example. It may also be useful, in certain types of analyses, such as pharmacokinetic and biodistribution studies, to label both the cationic and neutral components of the lipid carrier. Without some kind of tag, however, the process of detection may be more complex because of the need to efficiently extract the carrier lipid(s) from cells or tissues prior to analysis. The presence of endogenous lipids may make this difficult. For cationic lipids, such as DOTAP or DODAC, for example, it may be recommended that quantification of tissue levels, such as by HPLC analysis, be performed by those specializing in lipid analysis (Northern Lipids, Vancouver, BC, Canada). 

Products of Lipid Peroxide Decomposition. In view of the potential problems with direct lipid analysis, a simpler and more sensitive assay, the detection of thiobarbituric acid-reacting materials (TBARM), was used to detect DPE injury to membranes... 

Phospholipid fatty acid analysis (PLFA) is based on the determination of signature lipid biomarkers from the cell membranes and walls of microorganisms. Phospholipids are an essential part of intact cell membranes, and information from the lipid analysis provides quantitative insight into three important attributes of microbial communities viable biomass, community structure, and nutritional status. Phospholipid fatty acid prohles have been used to show the response of the microbial community to phosphorus availability (Keinanen et ah, 2002). Signature lipid biomarker analysis may not detect every species of microorganism in an environmental sample accurately, because many species have similar PLFA patterns. 

Several components of chlordane trans- and c/s-chlordane, trans- and c/s-nonachlor, heptachlor, gamma-chlordene) were detected in the skin lipids of humans (Sasaki et al. 1991b). The samples were taken by swabbing the face with cotton soaked with 70% ethanol 3-4 hours after the face was washed with soap. Because all samples from inhabitants of an area known to be contaminated with chlordane contained chlordane residues, and because the profile of chlordane components in skin lipids closely resembled those in technical chlordane, the authors suggested that skin lipid analysis is a satisfactory indicator of dermal exposure to airborne chlordane, such as occurs in homes treated for termites. Oxychlordane in the skin lipids was positively correlated (correlation coefficient = 0.68, p<0.01) with concentrations in internal adipose tissue. The authors concluded that the concentration of oxychlordane in skin lipids was a satisfactory indicator of body accumulation of chlordane. 

Thin-layer chromatography-flame ionization detection for lipid analysis... 

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]. 

The universal TLC facilities are utilized plates, adsorbents, microcapillaries, or micropipettes for sample application, development tanks, detection spray reagents, devices for spraying, and densitometers for quantification. Plates are either commercially precoated or handmade. Silica gel G (G, for gypsum as a binding substance), silica gel H (no binding substance) and, rarely, alumina and kieselguhr, form the thin-layer stationary phases. Complete sets of devices necessary for the preparation of handmade plates are commercially available. After the silica gel slurry is spread on the plates, they are left to dry in the air for at least 24 hr and shortly in an oven at 110°C. The plates are then ready for either direct use or for modification of the layer. From the great variety of precoated plates, which are commercially available and preferred nowadays, silica gel plates and plates with layers modified with carbon chains from C2 to C18 are of interest in lipid analysis. Understandably, precoated plates for Ag-TLC are not commercially available. Preparative TLC is performed mostly on 20 X 20 cm plates with layer thickness of... 

Regarding detection limits, both approaches [30,33] provided comparable results (about 400 pmol) and, therefore, both methods might be useful for routine lipid analysis—at least when major changes of the lipid compositions are expected. 

An example of DESI MS imaging of pharmaceutical components is presented in Fig. 1.5 (67). Lung tissues were collected from animals 30 min after dosing with clozapine. A tissue section was imaged using DESI MS across the w/z range of 200-1,100 to evaluate the sample for clozapine distribution and its metabolites. A variety of lipids were detected as well as clozapine and its 27-desmethyl metabolite. This analysis and the results from LC-MS/MS analysis performed in a complementary study confirmed the presence of clozapine in the lung. 

With a better idea of how to identify possible isobars at a given m/z value, a better characterization of the ions detected can be accomplished. The identification of the PLs for the tissue section analyzed is described in Table 12.3. As can be seen, there is a DHB cluster ion detected at every mass isolated (typically arising from a neutral loss of 137, 154, 155, or 176). Many of the ions isolated for tandem MS show three and even four lipid species. The three primary lipid classes detected in the positive ion mode were PC, PS, and PE. Cerebrosides were also detected, but were present at a much lower intensity due to the conditions employed (18). The primary reason for maintaining an isolation width of 1.5 for most experiments was that PLs are considered fragile ions in ion trap analysis (26, 31) and thus can be lost during isolation if the isolation width is not wide enough. 

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