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 f...