reference standard for quantification of BMG and BDG. A pure crystalline ethyl anthranilate azo derivative of UCB (AZO. integrated peak areas to concentrations of pure crystalline UCB. Quantification of UCB was carried out by using a standard curve relating a range of h.p.l.c. of their respective ethyl anthranilate azo derivatives. Bile pigments were eluted in order of decreasing polarity (glucuronide greater than glucose greater than xylose conjugates greater than UCB) and were chemically identified by t.l.c. Bile pigments were eluted with ammonium acetate buffer, pH 4.5, and a 20 min linear gradient of 60-100% (v/v) methanol at a flow rate of 2.0 ml/min for the preparative separations and 1.0 ml/min for the analytical separations. We employed an Altex ultrasphere ODS column in the preparative steps and a Waters mu-Bondapak C18 column in the separatory and analytical procedures. The method can be ‘scaled up’ for preparative isolation of pure BDG and BMG from pigment-enriched biles. method for analytical separation of biliary bile pigments and direct quantification of unconjugated bilirubin (UCB) and its monoglucuronide (BMG) and diglucuronide (BDG) conjugates in bile. We describe a facile and sensitive reverse-phase h.p.l.c.
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