Mass spectroscopy Carotenoids

Rivera-PastarnaD, YahiaEM and Gonzalez-Aguilar G. 2009. Identification and quantification of carotenoids and phenolic compounds in papaya using mass spectroscopy. In preparation. 

Carotenoids are generally light sensitive, easily oxidised by air and are affected by traces of acid, e.g. in solvents. These cause the polyenes to bleach or polymerize. The necessary precautions are therefore required to minimize these effects during isolation, purification and storage. They are identified by their UV-VIS spectra, and their molar extinction coefficients at specific wavelengths ( , max) have been used for characterisation and for quantitation. More recently ORD, CD, NMR, IR and mass spectroscopy have been used extensively. 

The mode of action of file phenylthiazolines has been investigated by looking for file accumulation of metabolic intermediates in chlorotic cress leaves. Treated plants showed an increase in levels of a compound that can not be observed in controls, identified by UV spectroscopy and HPLC cochromatography with an authentic phytoene. The latter has been produced by the application of file known carotenoid synthesis inhibitors such as flurtamon and identified independently by UV and mass spectroscopy. From these results, it can be assumed that chlorosis is caused by inhibition of carotenoid synthesis at the phytoene desaturase step. 

Elucidation of the structure of carotenoids requires, in addition to VIS/UV spectrophotometry, supplemental data from mass spectrometry and IR spectroscopy. Carotenoids are determined photometrically with high sensitivity based on their high molar absorbancy coefficients. This is often used for simultaneous qualitative and quantitative analysis. New separation methods based on high performance liquid chromatography have also proved advantageous for the qualitative and quantitative analysis of carotenoids present as a highly complex mixture in food. 

Other spectroscopic methods such as infrared (ir), and nuclear magnetic resonance (nmr), circular dichroism (cd), and mass spectrometry (ms) are invaluable tools for identification and stmcture elucidation. Nmr spectroscopy allows for geometric assignment of the carbon—carbon double bonds, as well as relative stereochemistry of ring substituents. These spectroscopic methods coupled with traditional chemical derivatization techniques provide the framework by which new carotenoids are identified and characterized (16,17). 

Enzell, C.R. and Back, S., Mass spectrometry, in Carotenoids Spectroscopy, IB, Britton, G., Liaaen-Jensen, S., and Pfander, H., Eds., Birkhanser, Basel, 1995, 261. 

Keywords Carotenoids analysis sample preparation UV/Visible spectroscopy geometrical isomers optical isomers HPLC mass spectrometry nuclear magnetic resonance metabolites. 

Degraded Carotenoids Physical Methods Separation and Assay N.M.R. Spectroscopy Mass Spectrometry Chiroptical Methods Electronic Absorption Spectroscopy Infrared and Resonance Raman Spectroscopy Other Spectroscopic Techniques Miscellaneous Physical Chemistry Photoreceptor Pigments Biosynthesis and Metabolism Stereochemistry Enzyme Systems Inhibition and Regulation... 

Essays in the older hterature (42, 43) deahng with the purification and characterisation of carotenoids have been superseded by recent accounts of the application of HPLC to carotenoid analysis and separation (551) and of modern techniques of high field n.m.r. spectroscopy to elucidation of their structures (216). The mass spectra of several of the fungal carotenoids discussed above have been studied in detail by Liaaen-Jensen (217). 

Early methods for the determination of carotenoid structure relied on chemical analysis. Although many of these procedures are still valuable, more recently physical methods of analysis have played an increasingly important role. Techniques such as mass spectrometry, light absorption, and nmr spectroscopy have proved particularly valuable. At present, determination of the structure of a carotenoid involves both physical and chemical methods, and final confirmation of the structure involves total synthesis of the compound. 

Carotenoids have also been characterized by nuclear magnetic resonance (NMR), infrared (IR), and Raman spectroscopy as well as by x-ray analysis and mass spectrometry (6). Because of their low volatility and relatively high molecular weight, the C-40 carotenoids have not as yet been separated by gas chromatography. Currently, carotenoids are primarily isolated by liquid chromatography on solid supports. 

This chapter focuses on the extraction and handling of retinoids and carotenoids, their separation by various chromatographic techniques, and their detection and quantitation, primarily by absorption spectrophotometry, fluorescence, and mass spectrometry. A variety of other methods exist for their identification and characterization, including circular dichroism (333), infrared spectroscopy (334), resonance Raman spectroscopy (335), NMR spectroscopy (336), and x-ray crystallography (337). Although some of these procedures require substantial amounts of a retinoid or a carotenoid in an essentially pure form for study, others, such as resonance Raman spectroscopy, are extremely sensitive and can be used to detect the localization of carotenoids in single cells (338,339). 

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