PREPARATIVE AND ANALYTICAL SEPARATIONS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND CAPILLARY ELECTROPHORESIS OF THE PRODUCTS FROM THE REACTION OF INSULIN WITH EXO-3,6-EPOXY-1,2,3,6-TETRAHYDROPHTHALIC ANHYDRIDE

An anion-exchange high-performance liquid chromatographic method using a TSKgel DEAE-2SW column and zone electrophoresis using a fused-silic a capillary are described for the preparative and analytical separatio ns of the products of the reaction between insulin and exo-3,6-epoxy-1 ,2,3,6-tetrahydrophthalic anhydride (ETPA), an amino protective reagen t. Four stable products were identified having exo-3,6-epoxy-1,2,3,6-t etrahydrophthalyl group(s) on insulin at the i) Gly(A1), ii) Gly(A1) a nd Phe(B1), iii) Gly(A1) and Lys(B29), and iv) Gly(A1), Phe(B1) and Ly s(B29) sites, together with four labile products produced by the react ion of ETPA with the non-amino groups of the stable products. The usef ulness of capillary electrophoresis was demonstrated in the evaluation of the effect of the molar ratios of anhydride to insulin on the prod uction of the insulin derivatives.