LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY OF CAROTENOIDS USING ATMOSPHERIC-PRESSURE CHEMICAL-IONIZATION

Carotenoids contained in plant extracts were analyzed using liquid chr omatography/positive-and negative-ion atmospheric pressure chemical io nization mass spectrometry (LC/APCI-MS) with a narrow-bore C-30 revers ed-phase high-performance liquid chromatographic (HPLC) column and a g radient solvent system containing methanol-methyl tert-butyl ether-amm onium acetate at a flow rate of 300 mu l min(-1). In addition to mass spectrometric detection, photodiode-array UV/visible absorbance detect ion was used between the HPLC column and mass spectrometer for additio nal carotenoid characterization. Positive-ion APCI produced protonated molecules and molecular ions for both xanthophylls and, unexpectedly, hydrocarbon carotenes; and during negative-ion APCI, M(-.) and [M - H ](-) ions were observed. In order to investigate the origin of the une xpected [M + H](+) ions, positive-ion APCI of beta-carotene was invest igated using deuterochloroform as the only solvent. Because beta-carot ene formed primarily deuterated ions, [M + D](+), during APCI in deute rochloroform, the mobile phase was determined to be the source of hydr ogen for protonation. The hydroxylated xanthophyll lutein fragmented d uring positive-ion APCI to eliminate water from the protonated molecul e and form the base peak of m/z 551. Using collision-induced dissociat ion in the ion source, additional fragmentation pathways characteristi c of tandem mass spectra of carotenoids were observed such as retro-Di els-Alder fragmentation, [M - 56](+.), for alpha-carotene and loss of toluene from the molecular ion, [M - 92](+.) for lutein, alpha- and be ta-carotene. The limits of detection for protonated molecules of alpha -carotene and lutein were approximately 3 and 13 pmol, respectively. I n negative-ion APCI, the limits of detection were approximately 3 and 1 pmol for M(-.) ions of alpha-carotene and lutein, respectively.