Charged chelate—capillary electrophoresis of endogenous corticosteroids

Abstract

Separation of endogenous 17‐ or 18‐hydroxylated corticosteroids (of the 21‐hydroxylated 4‐pregnen series) as charged chelates in capillary electrophoresis with borate as the ligand is demonstrated. Aldosterone, 18‐hydroxycorticosterone, 18‐hydroxy‐11‐deoxycorticosterone, cortisone, cortisol, and 11‐deoxycortisol are separated and resolved by 400 mM borate buffer at pH 9.0. Separation characteristics of the corticosteroid charged chelates were examined by varying the separation buffer borate concentration, pH, ionic strength, and addition of organic modifiers. The borate ion [B(OH)₄]⁻ is identified as the critical buffer component. Corticosteroids chelate borate with proximal hydroxyls composed of either the 17‐ or 18‐hydroxyl in combination with the 21‐position hydroxyl. Corticosteroid/borate chelation as indicated by CE results is corroborated with ¹¹B‐nuclear magnetic resonance (¹¹B‐NMR) sprectra. Chelation is a readily reversible process, with the strength of the resultant chelate, as opposed to the charge‐to‐mass ratio, predominantly determining analyte mobility in charged chelate ‐ capillary electrophoresis (CC‐CE).