Analysis of Carotenoids Using LC-MS-MS with Ion Mobility Spectrometry and Photoionization

Carotenoids are naturally occurring fat-soluble pigments found principally in bacteria and plants including edible plants. Because of their extensively conjugated carbon-carbon double bond system, carotenoids are potent antioxidants. The most abundant carotenoid and best singlet oxygen quencher found in red tomatoes is lycopene. Using lycopene as a potential dietary chemopreventive agent, a four-arm randomized, placebo-controlled, and double-blind phase II clinical intervention trial was carried out at the University of Illinois at Chicago. In the investigation, it was found that lycopene supplements at either 30 mg/d or 60 mg/d lycopene for 21 d were well tolerated by healthy men enrolled to the two lycopene intervention arms. Similar increases in serum levels of lycopene were observed in both treatment groups during this intervention study. However, stronger effects were observed for the two antioxidant biomarkers (urinary 8-oxo-deoxyguanosine and 8-iso-prostaglandin-F2) with the high dose intervention arm compared with its placebo control. Even though significant progress has been made in analyzing carotenoids, there is a continuing demand for more efficient and more sensitive analytical methods. Technologies that have seldom been applied to carotenoid analysis including ion mobility spectrometry and photoionization mass spectrometry were explored for their potential utility in this area. The use of LC-IM-tandem mass spectrometry helped confirm that in-source isomerization of lycopene, β-carotene, lutein, and zeaxanthin occurred during positive ion electrospray. This in-source isomerization was found to be temperature dependent. After separation using IM, the cis and all-trans isomers of lycopene, β-carotene, lutein, and zeaxanthin were shown by MS-MS with CID to produce unique fragmentation patterns that could be used to distinguish them. Atmospheric pressure photoionization is a mass spectrometric ionization technique that is new in carotenoid analysis. In the study, we first reported that carotenoids can be ionized during negative ion APPI. It was also suggested that dopants were essential for the formation of positive carotenoid ions as well as negative ions during APPI. Among the dopants evaluated, methyl-tert-butyl ether was found to be particularly effective towards the photoionization of carotenoids during negative ion APPI. As for positive ion APPI, the dopants anisole and chlorobenzene were the most effective.