3.3.2. Isolation of Alloxanthin from Aquatic Animals

According to our routine methods, carotenoid was extracted with acetone from animal tissue. The extract was partitioned between Et2O– *n*-hexane (1:1) and water in separating funnel. The organic phase was evaporated and saponified with 5% KOH/MeOH at room temperature for 2 h. Then, unsaponifiable compounds were extracted with Et2O– *n*-hexane (1:1, v/v) from the reaction mixture by addition of water. The organic layer was dried over Na2SO4 and evaporated. The residue was subjected to silica gel column chromatography increasing percentage of Et2O in *n*hexane. The fraction eluted with Et2O was subjected to HPLC on silica gel with acetone– *n*-hexane (3:7) to afford alloxanthin. Purity of alloxanthin was checked by UV-Vis, 1H-NMR, and MS spectral data. Then alloxanthin obtained from aquatic animals was subject to configurational analysis using a chiral column described above. 

## **4. Conclusions**

In conclusion, we synthesized stereoisomers of alloxanthin (**1a** –**<sup>c</sup>**) and established a HPLC analytical method using a chiral column to identify them for naturally occurring alloxanthin. Application of this method to various alloxanthin specimens of aquatic animals demonstrated that those isolated from shellfishes, tunicates, and crucian carp are identical with (3 *<sup>R</sup>*,3<sup>ȝ</sup>*R*)-stereoisomer **1a**, and unexpectedly those from lake shrimp, catfish, biwa goby, and biwa trout are mixtures of three  stereoisomers of **1a** –**<sup>c</sup>**. This is the first report of the occurrence of (3*S*,3<sup>ȝ</sup>*S*) and *meso*alloxanthin in nature. The analytical method can be a powerful tool to identify stereoisomers of alloxanthin in nature in a straightforward manner. 
