METHOD FOR REMOVAL OF SURFACE-ACTIVE IMPURITIES AND CALCIUM FROM CONJUGATED BILE-SALT PREPARATIONS - COMPARISON WITH SILICIC-ACID CHROMATOGRAPHY

Some commercial preparations of common natural conjugated bile salts c ontain impurities (e.g., amines, lipids, and calcium) that are likely to affect their physicochemical properties. A method was as developed for purifying commercial preparations of sodium salts of glycine- and taurine-conjugated bile acids. The method consists of passage of a dil ute aqueous solution of the sodium bile salt through three columns in sequence: graphitized carbon, a hydrophobic bonded octadecylsilane (C1 8) cartridge, and a calcium-chelating resin. The final solution was ex tracted with chloroform, and the purified bile salt was as then isolat ed by freeze-drying, with a yield of 65-75%. Each bile salt purified b y this method was compared with the corresponding bile salt purified b y conventional adsorption chromatography on a silicic acid column, usi ng a mixture of methanol and chloroform as eluant. Purity was assessed by visible spectra, by surface tension measurements (using the maximu m bubble-pressure method and a Wilhelmy wire method), by chloroform ex tractability of impurities in the conjugated bile acid, by liposome so lubilization, and by chemical analysis of the calcium content. Both pu rification methods removed colored and surface-active impurities, but the new method was always as or more effective than silicic acid colum n chromatography. Calcium ion, present in commercial bile salts in con centrations up to 16 mmol/mol bile salt, was removed completely by the three-column method, but not by silicic acid chromatography. The new method is thus a simple, rapid, and efficient procedure for purificati on of the sodium salts of glycine- and taurine-conjugated bile acids f or physicochemical measurements, in which elimination of surface-activ e impurities and polyvalent cations is desired.