Studies on steroids. CCXXXIII. Separation and characterization of bile acid disulphates in human urine by high-performance liquid chromatography.

The separation of disulphates of cholate, chenodeoxycholate, deoxycholate and ursodeoxycholate and their glyco and tauro conjugates has been carried out by high-performance liquid chromatography (HPLC) on a reversed-phase column. The chromatographic behaviour of bile acid disulphates was dependent on the position of the sulphate and hydroxy groups and the structure of the side-chain. The method has been applied to the separation and characterization of disulphates in biological fluids without prior deconjugation. The disulphate fraction was obtained from a urine specimen by passing it through a Sep-Pak C18 cartridge, followed by group separation by ion-exchange chromatography on a lipophilic gel, piperidinohydroxypropyl-Sephadex LH-20. Subsequent resolution into individual disulphates was attained by HPLC on Radial-Pak A and Cosmosil 5C18 columns. Taurochenodeoxycholate 3,7-disulphate and taurodeoxycholate 3,12-disulphate in human urine were unequivocally identified on the basis of their behavior in HPLC using mobile phases of different pH.