Reverse-phase h.p.l.c. separation, quantification and preparation of bilirubin and its conjugates from native bile. Quantitative analysis of the intact tetrapyrroles based on h.p.l.c. of their ethyl anthranilate azo derivatives

Abstract

A facile and sensitive reverse-phase HPCL [high-performance liquid chromatography] method for the analytical separation of biliary bile pigments and direct quantification of unconjugated bilirubin (UCB) and its monoglucuronide (BMG) and diglucuronide (BDG) conjugates in bile is described. The method can be scaled up for preparative isolation of pure BDG and BMG from pigment-enriched biles. An Altex ultrasphere ODS column was employed in the preparative steps and a Waters .mu.-Bondapak C18 column in the separatory and analytical procedures. Bile pigments were eluted with ammonium acetate buffer, pH 4.5, and a 20 min linear gradient of 60-100% (vol/vol) methanol at a flow rate of 2.0 ml/min for the preparative separations and 1.0 ml/min for the analytical separations. Bile pigments were eluted in order of decreasing polarity (glucuronide > glucose > xylose conjugates > UCB) and were chemically identified by TLC of their respective ethyl anthranilate azo derivatives. Quantification of UCB was carried out by using a standard curve relating a range of HPCL integrated peak areas to concentrations of pure crystalline UCB. A pure crystalline ethyl anthranilate azo derivative of UCB (AZO.cntdot.UCB) was employed as a single HPLC reference standard for quantification of BMG and BDG. Separation and quantification of biliary bile pigments are rapid (.apprx. 25 min) and bile pigment concentrations ranging from 1-500 .mu.M can be determined on line by using 5 .mu.l of bile without sample pretreatment. Bilirubin conjugates can be obtained preparatively in mg quantities without degradation or contamination by other components of bile. HPLC analyses of a series of mammalian biles show that biliary UCB concentrations generally range from 1-17 .mu.M. These values are considerably lower than those estimated previously by TLC, BMG is the predominant, if not exclusive, bilirubin conjugate in the biles of a number of rodents (guinea pig, hamster, mouse, prairie dog) that are experimental models of both pigment and cholesterol gallstone formation. Conjugated bilirubins in the biles of other animals (human, monkey, pony, cat, rat and dog) are chemically more diverse and include mono-, di- and mixed diconjugates of glucuronic acid, xylose and glucose in proportions that give distinct patterns for each species.