The unusual gradient elution for reversed phase HPLC of a strong chelator as an active drug substance.

The new approach to drug development, especially for cardiovascular and brain diseases, brought to synthesis of new lipophillic derivatives of strong calcium chelator BAPTA - DP-b99 and DP-109. Due to their chelating ability, these compounds require metal-free stationary phases, and their high hydrophobicity resulted in unusually steep gradient elution. Novel HPLC methods for analysis of these two compounds were developed. Purospher RP-C18, 5 microm, 125 x 3.0 mm and XTerra RP18, 3.5 microm, 100 x 4.6 mm columns with a steep gradient from: 1% acetic acid to acetonitrile were used for DP-b99, and Hypersil HyPurity C4, 5 microm, 100 x 4.6 mm column with a steep gradient from 1% Acetic acid to 5% THF in methanol -- for DP-109. Versatile detection techniques could be used with these LC procedures. The methods appeared to be sensitive, selective, reproducible and stability indicating. They could be easily upgraded to bioanalytical methods with LC-MS technique.