1987 Volume 36 Issue 6 Pages 352-355
A simple and rapid method for fractionating serum bilirubin by means of HPLC has been established by modifying the method of Lauff et al. In our procedure, the cumbersome and time-consuming pretreatment of serum in the previous method is replaced by the use of the guard column (Hiber Lichro CART 4-4, Merck). This guard column retains high-molecular weight serum proteins to protect the analytical column from the irreversible adsorption, and it is changed every 20 samples or whenever the tailing is observed in the chromatogram. The present method requires only the dilution of 50μl of serum with 500μl of sodium chlorate solution (0.2w/v% sodium chlorate and 0.05v/v% Triton X-100 in 0.5M phosphate buffer, pH 6.8), and subsequent filtration of the mixture through a 0.45μm Millipore filter. Ten microliters of the filtrate is injected into the column; the HPLC conditions are the same as described by Lauff. The four fractions of serum bilirubin are obtained. The relative standard deviations for each bilirubin concentration in the fractions are 5.4 to 16.7% (n=5). The correlation coefficients with other methods including the Lauff's method are 0.863 to 0.974.
