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

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