CONTINUOUS-FLOW FAST-ATOM-BOMBARDMENT LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY OF CAROTENOIDS

High-performance liquid chromatography (HPLC) coupled with UV/visible absorbance detection is currently the method of choice for the separation and identification of thermally labile and nonvolatile carotenoids. However, UV/visible absorption data used in combination with HPLC retention characteristics permit only tentative identification of carotenoids. In order to identify carotenoids eluting from chromatographic columns more rigorously and to obtain data complementary to absorbance spectroscopy, carotenoid molecular ions were identified using continuous-flow fast atom bombardment (FAB) liquid chromatography/mass spectrometry (LC/MS) with coaxial flow addition of the FAB matrix, 3-nitrobenzyl alcohol. The limit of detection of this LC/MS technique was determined to be approximately 5 ng for lutein and 15 ng for alpha-carotene, compared to 40 and 70 ng for lutein and alpha-carotene, respectively, using a photodiode array detector. In addition to molecular weight confirmation, structurally significant fragment ions specifically identifying the isomeric carotenoids, alpha-carotene, beta-carotene, and lycopene, were obtained during LC/MS using the MS/MS technique of B/E linked scanning with collisional activation. Finally, continuous-flow FAB LC/MS with coaxial-flow matrix addition was used to identify carotenoids present in a tomato extract. Carotenoid molecular ions were detected at m/z 536, 542, 544, and 540 corresponding to lycopene and beta-carotene (both weighing 536), phytofluene, phytoene, and two isomers of zeta-carotene, respectively.