High-performance ion-pair chromatographic behaviour of conjugated bile acids with di-n-butylamine acetate

This paper dealt with a simple and efficient method for separating a mixture of different series of ionic, high polar, and hydrophilic conjugates of bile acids by high-performance ion-pair chromatography (HPIPC) with a new volatile ion-pair chromatographic reagent, di-n-butylamine acetate (DBAA), as a mobile phase additive. The substrates examined included eleven different classes of C-24 glycine- or taurine-amidated, 3-sulfated, 3-glucosylated, 3-N-acetylglucosaminidated, and 3-glucuronidated conjugates of cholic, chenodeoxycholic, urosodeoxycholic, and deoxycholic acids, as well as their double-conjugated forms. The anionic conjugated bile acids were chromatographed on a C18 reversed-phase ion-pair column, eluting with methanol–water (65:35, v/v) containing 5 mM of DBAA as a counter ion. Satisfactory chromatographic separation and column performance were attained by DBAA, compared with conventionally used non-volatile tetra-n-butylammonium phosphate. The present HPIPC method with DBAA provides an insight into the separation and structural elucidation of these biologically important bile acid conjugates and may be proved to be applied to HPLC–mass spectrometric analysis.