6.1.4Current Studies and Possible Structures of Higher Molecular Weight Products

Fig. 6.5 The mass spectrum of the Folch extract of human lipofuscin at time 62.93 and 86.26 min (inset). Group I, II, and III identify the related clusters of higher molecular weight compounds with mass to charges of approximately 800, 1,000, and 1,400 respectively. The arrow indicates the addition of 14 amu to m/z 847.9

complex mixture. We have assumed that all molecules in the mixture have similar ionization efficiency, since all of the instrumental parameters, flow rate, and solvent composition remained constant. After further analysis of the mass spectral data of compounds that eluted from 50 to 100 min, the chromatogram revealed a series of closely related compounds that appeared to be related by a mass of 14 amu, which could be methylene groups. Figure 6.5 displays a representative mass spectrum of compounds that eluted at approximately 60 min with an inset spectrum of compounds eluting at 80 min. There are three clusters of eluting masses, labeled I, II, and III, eluting in the ranges of 800, 1,000, and 1,400 amu.

To determine if these components were structurally related to A2E, the tandem mass spectroscopy (MS/MS) data were analyzed. The MS/MS spectrum and the total absorption of A2E are displayed in Fig. 6.6a, b respectively. The fragmentation pattern displayed characteristic losses of 150, 174, and 190 from the parent ion mass of 592 amu. These distinctive cleavages are illustrated in Fig. 6.6c. Once identified, these losses were compared to the MS/MS data of the components located within the complex mixture of the lipofuscin sample and all of the material that eluted between 50 and 80 min and approximately 50% of the material between 80 and 110 min had analogous spectra exhibiting fragments with the loss of the same m/z fragments.

Figure 6.7a, b display the MS/MS and absorption spectrum of peak with M+ 858, which is representative of components that eluted at approximately 60 min

b

Fig. 6.6 (a) The MS/MS scan for A2E identified in the Folch extract of Lipofuscin granules. Peaks corresponding to the mass of 592 with the loss of 106, 150, 174, and 190 are identified. (b) The UV-visible spectrum of A2E. (c) Characteristic cleavages for the fragmentation of A2E

Fig. 6.7 (continued)

displayed in Fig. 6.4. Once analyzed, Fig. 6.7a displayed ions with masses of 752, 708, 683, and 668, which correspond to losses of 106, 150, 174, and 190 from the parent ion of 858. The peak with m/z 752 is from the loss of xylene, which is commonly observed in polyene compounds that have more than four conjugated double bonds [58, 59]. The absorption spectra in Fig. 6.7b shows two maxima at 330 and 500 nm in the lipofuscin and aged A2E samples. The proposed structure for this 500 nm absorbing species is displayed in Fig. 6.7c. Previously, Radu et al. reported that the 500 nm absorbing species was A2PE-H2 [60]. However, Fishkin et al. later reported that this absorption was from an all trans retinal dimer conjugated to phosphatidylethanolamine (ATR dimer-PE) [61]. Following the Fishken study, Bui et al. further examined native retinal fluorophores involved in the formation of A2E including the 500 nm absorbing species. Based on the absorption and mass spectra, this study concluded that A2E-H2 is not only a precursor to the synthesis of A2E but is also the compound responsible for the 500 nm absorbing species in lipofuscin [62]. However, the fragmentation pattern and absorption spectrum reported here suggests that the 500 nm absorbing species is an A2E derived higher molecular weight product.

The components that eluted with masses in the range of 1,000 and 1,400 amu were analyzed, and the MS/MS data was again compared to the fragmentation pattern of A2E. Figure 6.8a and b present the MS/MS spectra for M+ 1,081 and 1,423, respectively. The fragmentation pattern for M+ 1,081 displayed ions with masses of

Fig. 6.8 (a) The MS/MS scan for M+ 1,081 located in lipofuscin. Peaks corresponding to the mass of 1,081 with the loss of 150 (m/z 931), 174 (m/z 907), and 190 (m/z 891) are identified. The mass of A2E (m/z 592) and additional peaks corresponding to smaller molecular weight compounds (m/z 813, 825, and 865) with similar losses identified in the same sample. Peaks corresponding to the mass of 1,081 with the loss if 92 (m/z 989) for toluene and the loss of 106 (m/z 975) for xylene are also identified. (b) The MS/MS results for the fragmentation of peak with M+ 1,423 in the lipofuscin sample. Peaks corresponding to the mass of 1,423 with the loss of 174 (m/z 1,249) and 190 (m/z 1,233) are identified. The mass of A2E (m/z 592) and additional peaks corresponding to smaller molecular weight compounds (m/z 757, 863, and 1,019) with similar losses identified in the same sample. Peaks corresponding to the mass of 1,423 with the loss of 92 (m/z 1,331) for toluene and the loss of 106 (m/z 1,317) for xylene are also identified