Carotenoids ionization

APCl in positive mode ionization and triple quadrupole detection was used for determination of free and bound carotenoids in paprika, obtaining the [M + H]+ and losses of fatty acids as neutral molecules from the [M + H]+ with MeOH, MTBE, and H2O as eluent from the C30 column. The positions of the fatty acids on unsymmetrical xanthophylls could not be established by the MS data. 

Li, H. et ah. Determination of carotenoids and all-fra 5-retinol in fish eggs by liquid chromatography-electrospray ionization-tandem mass spectrometry, J. Chromatogr. B, 816, 49, 2005. 

Van Breemen, R.B. et al.. Liquid chromatography/mass spectrometry of carotenoids using atmospheric pressure chemical ionization, J. Mass Spectrom., 31, 975, 1996. Tian, Q., Duncan C.J.G., and Schwartz S. J., Atmospheric pressure chemical ionization mass spectrometry and in-source fragmentation of lutein esters, J. Mass Spectrom., 38, 990, 2003. 

This review will first concentrate on the unimolecular gas-phase chemistry of diene and polyene ions, mainly cationic but also anionic species, including some of their alicyclic and triply unsaturated isomers, where appropriate. Well-established methodology, such as electron ionization (El) and chemical ionization (Cl), combined with MS/MS techniques in particular cases will be discussed, but also some special techniques which offer further potential to distinguish isomers will be mentioned. On this basis, selected examples on the bimolecular gas-phase ion chemistry of dienes and polyenes will be presented in order to illustrate the great potential of this field for further fundamental and applied research. A special section of this chapter will be devoted to shed some light on the present knowledge concerning the gas-phase derivatization of dienes and polyenes. A further section compiles some selected aspects of mass spectrometry of terpenoids and carotenoids. 

Several original papers must be mentioned that deal with mass spectrometric techniques which the numerous reviews do not comprise. Kaufmann and coworkers268,288 studied the mass spectrometric analysis of carotenoids and some of their fatty acid esters using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and its post-source-decay (PSD) variant. Some advantages concerning the thermal instability and limited solubility were discussed, but the fragmentation paths of the carotenoid cations were found to be essentially the same as those observed with conventional techniques. 

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. 

ELECTRON IMPACT AND CHEMICAL IONIZATION MASS SPECTROMETRY OF CAROTENOIDS... 

Mass Spectrometry of Carotenoids NOTE For LC/MS or flow injection using continuous-flow FAB, the mass spectrometer must be equipped with a continuous-flow ionization source. 

When HPLC is used as part of the analysis, the mobile phase is typically a mixture of methanol and methyl-tert-butyl ether (i.e., 50 50, v/v), although other HPLC solvents for LC/MS using APCI (e.g., water, tetrahydrofuran) can be used. It is important to note that if combustible nonaqueous solvent systems are used, water or a halogenated solvent such as methylene chloride or chloroform should be added to the mobile phase postcolumn to suppress ignition in the ion source. In addition, the APCI source must be vented outside the laboratory and should not allow air into the ionization chamber. A scan range of m/z 300 to 1000 will include the known carotenoids and their most common esters. 

The fragmentation patterns and characteristic fragment ions for the carotenoids observed in FAB-MS and LSIMS tandem mass spectra are also observed in the tandem mass spectra obtained following ESI (see Basic Protocol 4), APCI (see Basic Protocol 5), and other methods. A detailed account of structure determination of carotenoids using FAB ionization with CID and MS/MS is presented in van Bree-men el al. (1995). Finally, another advantage of MS/MS is that matrix ions formed during FAB-MS or LSIMS, and any other contaminating ions, are eliminated, which simplifies interpretation of the mass spectrum. 

FAB ionization has been used in combination with LC/MS in a technique called continuous-flow FAB LC/MS (Schmitz et al., 1992 van Breemen et al., 1993). Although any standard HPLC solvent can be used, including methyl-ferf-butyl ether and methanol, the mobile phase should not contain nonvolatile additives such as phosphate or Tris buffers. Volatile buffers such as ammonium acetate are compatible at low concentrations (i.e., <10 mM). Continuous-flow FAB has also been used in combination with MS/MS (van Breemen et al., 1993). The main limitationsof continuous-flow FAB compared to other LC/MS techniques for carotenoids, such as ESI and APCI, are the low flow rates and the high maintenance requirements. During use, the 3-nitrobenzyl alcohol matrix polymerizes on the continuous-flow probe tip causing loss of sample signal. As a result, the continuous-flow probe must be removed and cleaned approximately every 3 hr. 

MALDI-TOF-MS facilitates the analysis of carotenoids and other natural products with detection limits that are lower than most other techniques. For example, subpicomole quantities can be detected (Wingerath et al., 1999). The enhanced sensitivity is the result of the efficiency of the pulsed ionization and detection system in which a complete mass spectrum is recorded with each laser flash. Like FAB and LSIMS, molecular ions are the most abundant sample ions, although some protonated molecules and [M-H]+ ions may be formed as well. Abundant molecular ions of carotenoid esters have been observed using MALDI-TOF-MS (Kaufmann et al., 1996 Wingerath et al., 1996),... 

The choice of matrix for FAB-MS, LSIMS, and MALDI is essential for efficient sample ionization. For example, the use of 3-nitroben-zyl alcohol instead of glycerol, thioglycerol, or most other more common matrices is essential for the formation of abundant carotenoid ions during FAB and LSIMS. Nonpolar carotenoids (e.g., the carotenes) are insoluble in polar ma-... 

Because extensive fragmentation is typical of El and Cl mass spectra, molecular ions or protonated molecules might not be observed. In order to confirm the molecular weight of a carotenoid, desorption El or desorption Cl (also known as in-beam El and Cl) can be utilized to increase the abundance of the molecular ion species. If the molecular weight of the carotenoid remains uncertain, then softer ionization techniques should be investigated, such as FAB-MS, ESI, MALDI, or APCI. 

The limits of detection for carotenoids using FAB-MS and LSIMS are not as low as with most other ionization techniques (Schmitz et al., 1992). Therefore, >10 pmol of each carotenoid should be loaded onto the direct insertion probe per analysis. The matrix, 3-nitrobenzyl alcohol, has been effective in facilitating the ionization of all types of carotenoids. However, more polar matrices such as glycerol or thioglycerol might be useful for the FAB-MS or LSIMS analysis of polar xanthophylls such as astaxanthin. Because glycerol and thioglycerol are poor solvents for hydrophobic compounds, they are unlikely to solvate and thus facilitate the ionization of the nonpolar carotenes such as (3-carotene. 

Electrospray ionization will produce molecular ions, M+, with almost no fragmentation for carotenes and many xanthophylls. As the polarity of the carotenoid increases, the prob-... 

Kaufmann, R., Wingerath, T., Kirsch, D., Stahl, W., and Sies, H. 1996. Analysis of carotenoids and carotenol fatty acid esters by matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source-decay mass spectrometry. Anal. Biochem. 238 117-128. 

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