Simultaneous HPLC Determination of Free, Conjugated, and Sulfated Bile Acids

Abstract

An HPLC method has been developed to separate the 18 major bile acids in a single analytical run (cholate, chenodeoxycholate, deoxycholate, lithocholate, sulfolithocholate, ursodeoxycholate and their glycine and taurine conjugates). Bile acids are rapidly extracted from bile, urine or peritoneal fluid over a C18 cartridge and resolved on a reverse-phase column. Peaks at 210 run are quantitated by comparison with the peak area of commercial standards using 7α, 12α-dihydroxycholanic acid as a retention standard. Standard regression lines are linear over the range of 2–40 μg for conjugated acids and 50–800 μg for unconjugated acids (correlation coefficient range, r=0.962 to 0.989; coefficient of variation, CV=7.8%). Limits of detection are 1 and 20 μg respectively. In a preliminary evaluation, the analysis of urine demonstrated profile shifts in conjugation pattern and total bile acid concentration between patients with hepatitis, biliary obstruction and nonhepatic illness. Abnormal urine profiles from another patient with chronic cholecystitis changed toward a normal pattern in the weeks post-cholecystectomy. This method can thus provide a rapid, sensitive means of analyzing bile acid profiles in a variety of body fluids.