Abstract
Nine chlorophyll derivatives from different spinach preparations were separated and identified using reversed-phase high-performance liquid chromatography—mass spectrometry (HPLC—MS). These pigments included chlorophylls a and b, chlorophyllides a and b, pheophorbide a, pheophytins a and b, and pyropheophytins a and b. HPLC—MS measurements were carried out using HPLC—frit-fast atom bombardment (FAB)-MS, which is a continuous-flow FAB-MS interface. The highly hydrophobic chlorophyll derivatives were eluted from a reversed-phase HPLC column using a gradient of increasing ethyl acetate concentration. Glycerol was included in the mobile phase to serve as the matrix for FAB ionization. During analysis by positive-ion HPLC—frit-FAB-MS, abundant protonated molecules, [M + H] + were detected for all nine chlorophyll derivatives. Fragment ions were observed in the mass spectra that were similar to those produced during standard probe FAB-MS. These HPLC—MS procedures were shown to be useful for the rapid separation and identification of a variety of chlorophyll derivatives from natural sources.
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