IDENTIFICATION OF CHLOROPHYLL DERIVATIVES BY MASS-SPECTROMETRY

Chlorophylls a and b and the corresponding pheophytins, chlorophyllides, pheophorbides, and pyropheophytins were extracted from fresh spinach leaves or heat-treated spinach tissue, and zinc pheophytin a and zinc pyropheophytin a were prepared from pheophytin a and pyropheophytin a, respectively. Following separation using either reversed- or normal-phase HPLC, the molecular weight of each chlorophyll derivative was confirmed by positive ion fast atom bombardment (FAB) mass spectrometry. The isotope pattern of each molecular ion indicated whether or not the chlorophyll derivative contained magnesium or zinc. Next, tandem mass spectrometry (MS/MS) following collisional activation was used to obtain structural information that characterized each class of chlorophyll with respect to the presence or absence of the phytyl chain and the beta-keto ester group on C-10. These HPLC and mass spectrometric data will serve as useful references for the isolation and structural determination of chlorophyll derivatives from synthetic or natural sources.