Reversed-phase liquid chromatography of biologically active lipophilic chelators. II. Improvement of chromatographic performance and selected applications in biochemical analysis.

Lipophillic derivatives of strong calcium chelator BAPTA--DP-b99 and DP-109--are potential drug substances for cerebrovascular and neurodegenerative diseases. The previously published reversed phase HPLC methods for these compounds suffered from integration problems due to gradient dip, insufficient repeatability and peak shape. A C4 column rapidly aged. The addition of acetic acid to the organic part and of ammonium acetate to the aqueous part results in more symmetric peaks, improves method precision and solves integration problems. Washing the column in both directions with a combination of methanol, tetrahydrofuran and water extends its use. The improved methods are sensitive, selective, reproducible, and stability indicating. Impurities and degradation products were identified by LC-MS. Versatile detection techniques can be used with these HPLC methods, allowing performance of bioanalysis with mass spectrometric or flow scintillation detectors. The bioanalytical application of these methods is illustrated by examples of pharmacokinetic and metabolic studies with the labeled compounds.