Atmospheric-pressure chemical ionization lipid analysis

Atmospheric Pressure Chemical Ionization (APCI)/MS APCI/MS is used to analyze compounds of intermediate molecular weight (100-1,500 da) and intermediate polarity and is particularly useful for the analysis of biochemicals such as triacylglycerides, carotenoids, and lipids (Byrdwell, 2001). For volatile, nonpolar compounds of low molecular weight, GC/MS is preferred to APCl/MS whereas APl-electrospray/ MS provides better results for larger, more polar materials. The selection of APCl/MS over GC/MS or APl-electrospray/MS depends on the compounds to be analyzed. Many LC/MS instruments can be easily switched between APCl/MS and APl-electrospray/MS so that it can be rapidly determined which ionization process is more suitable to a given chemical. Additional manipulations such as pre and postcolumn derivatization reactions (Nagy et al., 2004 Peters et al., 2004) or coulometric oxidation (Diehl et al., 2001) can make the chemicals of interest more amenable to detection by APCI. 

Byrdwell WC. 2001. Atmospheric pressure chemical ionization mass spectrometry for analysis of lipids. Lipids 36 327. 

Advanced analytical techniques, particularly mass spectrometry (MS), often combined with liquid chromatography (LC) or gas chromatography (GC), are requisite for lipid analysis and they have played the crucial role in the emergence as well as the progresses of lipidomics. MS is the principal choice for the lipid analysis, particularly using electrospray ionization (ESI) and sometimes also atmospheric pressure chemical ionization or laser-based MS methods for surface analysis. The MS-based techniques are the best choice for lipidomics due to their superior sensitivity and molecular specificity, and because they provide the ability to resolve the extensive compositional and structural diversity of lipids in biological systems. 

In recent years the rapid development of high-sensitivity analytical techniques such as mass spectrometry (MS) and liquid chromatography (LC) supported the investigation of the endocannabinoids as part of a complex lipid network. The identification of lipid components of the endocannabinoid system can be achieved in a single analytical step by state-of-the-art platforms such as tandem mass spectrometry (MS/MS), which provides the detailed structural information necessary for characterization of lipids and increases specificity in complex biological matrices. Furthermore, the implementation of ionization techniques such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) allow the coupling of LC to MS, and permits the separation and analysis of endocannabinoids with greater speed and accuracy. 

Byrdwell, W.C. and Emken, E.A. (1995) Analysis of triglycerides using atmospheric-pressure chemical ionization mass-spectrometry. Lipids, 30(2), 173-175. 

Pajkovic, N. van Breemen, R. 2005. Analysis of carotenoids using atmospheric pressure chemical ionization mass spectrometry. In Modem Methods for Lipid Analysis by Liquid Chromatography (Byrdwell, C., Ed.). AOCS Publishing, Champaign, IL, pp. 413 30. 

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... 

Laakso P, Voutilainen P. Analysis of triacylglycerols by silver-ion high-performance liquid chromatography-atmospheric pressure chemical ionization spectrometry. Lipids 1996 31 1311-22. 

Nearly all known ionization methods of mass spectrometry (including electron impact, laser desorption and fast atom bombardment) were already successfully applied to lipids. However, many ionization techniques are not very suitable for the analysis of complex PL mixtures as they provide considerable amounts of fragment ions. Therefore, only three soft-ionization methods play nowadays a major role in lipid analysis. Beside atmospheric pressure chemical ionization (APCI) (Byrdwell 2001), electrospray ionization (ESI) (Pulfer and Murphy... 

One of the most powerful techniques used in Upid analysis today is HPLC coupled with mass spectrometry (HPLC/MS). Several mass spectrometric ionization techniques, such as fast atom bombardment (FAB) [23], electrospray ionization (ESI) [29,30], ionspray ionization (ISI) [31], and atmospheric pressure chemical ionization (APCI) [22,30,32] have been used. By using HPLC/MS, one can get information on the molecular structure of the intact lipids, which helps differentiate molecular species within different lipid classes. By using tandem mass spectrometry (MS/MS), identification of molecular species of different sphingolipids can be achieved in an easier and more sensitive way. There are many other advantages of using MS, such as small sample size, minimal sample preparation, and lack of need for derivatization, speeds, and sensitivity. In the literature, sphingolipids of both animal and plant origin were analyzed by MS. 

The content of this chapter focuses on the analysis of intact polar lipids by high-pressure liquid chromatography (HPLC) with flow or loop injection -and mass spectrometry (MS) or tandem mass spectrometry (MS-MS) using thermospray (TS), discharge-assisted TS [or plasmaspray (PSP)], electrospray (ES) and atmospheric pressure chemical ionization (APCI). It was intended to include only those papers describing the analysis of intact polar lipids by liquid chromatography on-line with MS. However, many papers describe flow or loop injection with MS and/or the analysis of derivatives of polar lipids. These papers, describing excellent applications of MS and/or MS-MS of polar lipids, are included since this chapter would not have been complete without them. 

In Chapter 9 Karlsson explains the importance of combined techniques, for example joint use of HPLC, flow or loop injection and MS or tandem MS in the analysis of lipids. The techniques of thermospray, electrospray and atmospheric pressure chemical ionization are described. Applications range from platelet activating factor to cardiolipin. Karlsson mentions some of the practical procedures which he adopts in his laboratory to ensure good reproducible analysis. 

In principle however, this method should be portable to other mass spectrometer systems capable of chemical ionization and tandem mass spectrometry. An [M+54] ion was reported using atmospheric pressure chemical ionization (APCI) with a predominantly acetonitrile solvent while analyzing extremely long-chain polyunsaturated fatty acids (16). Such an observation is promising for the use of this method for the analysis of low- or nonvolatile lipids, such as triglycerides and phospholipids. 

Electrospray ionization and atmospheric pressure chemical ionization are popular as ionization techniques, for qualitative and quantitative LC—MS analysis of lipids [63—65]. Based on flieir ionization mechanisms, ESI is more suitable for ionization of polar and ionic compounds and is capable of ionizing both small and large biomolecules. APCI can ionize less polar and neutral compounds more efficiently than ESI. Consequently, APCI—MS coupled to LC is the most used tool for TAG identification, because of the full compatibility with common NARP LC conditions, easy ionization of nonpolar TAGs, and the attainment of both protonated molecules [M + H]+ and fragment ions [M - - H — RzCOOH]. On the other hand, ESI is usually employed for the more-polar phospholipids. However, ESI or matrix-assisted laser desorption—ionization (MALDI) have been used for TAGs, as well [66,67]. 

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. 

Atmospheric-pressure chemical ionization (APCI) has been used for analysis of relatively polar lipids in comparison to those analyzed by El and Cl. APCI/MS spectra are relatively simple, with a protonated molecular ion as the most common base peak. Many classes of lipids, including free fatty acids, phospholipids, sterols, and TG, are ionizable by APCI. Although APCI is a relatively soft ionization technique, the comparatively harsh conditions employed relative to electrospray ionization (ESI) still induce some degree of fragmentation. After they were made commercially avaOable in the late 1980s and early 1990s, both ESI and MALDI mass spectrometers were extensively used for analysis of almost all nonvolatile lipids in their intact forms. Summaries for the majority of these studies can be found in multiple valuable review articles. " " ... 

Cai, S. S. Syace, J. A. Comparison of atmospheric pressure photoionization, atmospheric pressure chemical ionization, and electrospray ionization mass spectrometry for analysis of lipids. Anal. Chem. 2006, 78, 1191-1199. 

Multiple new developments related to the different components of mass spectrometers (e.g., ion source and analyzer) have been made in the last several years. For example, atmospheric-pressure photoionization (APPI) has become the newest family member of atmospheric-pressure ionization (API) techniques in addition to ESI and APCI. APPI has provided a new approach to analyze compounds notreadily ionized by ESI with improved ionization efficiency in comparison to APCI. Therefore, APPI serves as a complementary alternative to ESI for lipid analysis. Since APPI requires less heat for desolvation than APCI, thermally labile compounds can be analyzed with fewer concerns for thermal chemical alterations or degradation in the ionization source. In addition, APPI offers lower detection limits, higher signal intensities, and higher signal to noise ratios in comparison to APCI. Therefore, APPI has been well-appreciated for its ability to analyze many neutral lipid classes, including free fatty acids (and their esters), MG, DG, TG, sterols, fat-soluble vitamins, and even polar phospholipids. 

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