MEASUREMENT OF CONJUGATED AND UNCONJUGATED BILIRUBIN IN BILE .2. NEW THIN-LAYER CHROMATOGRAPHIC METHOD

Abstract

A new TLC method was described for measurement of conjugated (CB) and unconjugated (UCB) bilirubin in bile. Bile was streaked on a silica-gel plate and developed with chloroform. Bilirubin in the mobile UCB band and stationary CB band was quantitated by diazotization from the scraped gel with p-iodoaniline reagent. Both CB and UCB in bile yielded identical azopigment absorbance. There was no contamination of the UCB band with either CB or oxidative derivatives of bilirubin, and over 98% of added 14C-UBC migrated with the mobile band. Recoveries from TLC averaged 98.2 .+-. 4.3% of the applied pigment, duplicates agreed within 10%, and the method accurately detected the enzymatic and alkaline hydrolysis of CB to UCB. Concentrations of UCB in 9 samples of normal human gallbladder bile were less than 1.9 mg/dl and never exceeded 1.2% of the total bilirubin.