HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY CONTINUOUS-FLOW FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRY OF CHLOROPHYLL DERIVATIVES

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, pheophoride a, pheophytins a and b, and pyropheophytins a and b. PHLC-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.