ELECTROSPRAY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY OF CAROTENOIDS

The first method for electrospray liquid chromatography-mass spectrometry (LC-MS) of carotenoids is reported, which uses a C-30 reversed-phase HPLC column and a gradient solvent system containing methanol/methyl tert-butyl ether/ammonium acetate at a flow rate of 1.0 mL/ min. The entire HPLC column effluent passes through a photodiode away absorbance detector and then into the electrospray LC-MS interface without solvent splitting. In this way, maximum sensitivity is achieved for both the photodiode array detector, which records the UV/vis spectra of each carotenoid, and the mass spectrometer, which measures the molecular ions of each carotenoid. Molecular ions, M(.+), without evidence of any fragmentation, were observed in the electrospray mass spectra of both xanthophylls and carotenes. In order to enhance the formation of molecular ions, solution-phase carotenoid oxidation was carried out by means of postcolumn addition of a halogenated solvent to the HPLC effluent, Several different halogenated solvents were evaluated, including chloroform, 2,2,3,3,4,4,4-heptafluoro-1-butanol, 2,2,3,3,4,4,4-heptafluorobutyric acid, 1,1,1,3,3,3-hexanuoro-2-propanol, and trifluoroacetic acid. Among these halogenated solvents, 2,2,3,3,4,4,4-heptafluoro-1-butanol at a concentration of 0.1% (v/v) was found to produce the best combination of carotenoid molecular ion abundance and reproducibility. The limits of detection for lutein and beta-carotene were between 1 and 2 pmol each, which was 100-fold lower than the detection limit of the photodiode array detector signal.