Liquid Chromatography/Mass Spectrometry of Carotenoids Using Atmospheric Pressure Chemical Ionization

Abstract Carotenoids contained in plant extracts were analyzed using liquid chromatography/positive‐and negative‐ion atmospheric pressure chemical ionization mass spectrometry (LC/APCI‐MS) with a narrow‐bore C 30 reversed‐phase high‐performance liquid chromatographic (HPLC) column and a gradient solvent system containing methanol–methyl tert‐butyl ether–ammonium acetate at a flow rate of 300 μl min ‐1 . In addition to mass spectro‐metric detection, photodiode‐array UV/visible absorbance detection was used between the HPLC column and mass spectrometer for additional 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 unexpected [M+H] + ions, positive‐ion APCI of β‐carotene was investigated using deuterochloroform as the only solvent. Because β‐carotene formed primarily deuterated ions, [M+D] + , during APCI in deuterochloroform, the mobile phase was determined to be the source of hydrogen for protonation. The hydroxylated xanthophyll lutein fragmented during positive‐ion APCI to eliminate water from the protonated molecule and form the base peak of m / z 551. Using collision‐induced dissociation in the ion source, additional fragmentation pathways characteristic of tandem mass spectra of carotenoids were observed such as retro‐Diels–Alder fragmentation, [M‐56] +• , for α‐carotene and loss of toluene from the molecular ion, [M‐92] +• for lutein, α‐and β‐carotene. The limits of detection for protonated molecules of α‐carotene and lutein were approximately 3 and 13 pmol, respectively. In negative‐ion APCI, the limits of detection were approximately 3 and 1 pmol for M ‐• ions of α‐carotene and lutein, respectively.