[Using purified conjugated bilirubin as a calibrator for bilirubin measurement]

To improve the accuracy and precision of the determination of bilirubin, especially direct bilirubin (DB), and the standardization of that as well.

Purified conjugated bilirubin (Bc) and ditaurobilirubin(DTB) and their diazo products were subjected to absorption spectrum analysis. The diazo reaction characters of their calibration solutions were compared by the method of Doumas J-G(TB & DB).

Bc, DTB and their azopigments were found to have the similar absorption spectra with the same lambda max. Their TB standard curves almost superposed together all over. Although the slopes of their DB standard curves were not markedly different ((YBc = 0.00366X + 0.00933, rBc2 = 0.9977, P < 0.01; YDTB = 0.00391X + 0.00023, rDTB2 = 0.9987, P < 0.01; Pb1-b2 > 0.05, n1 = n2 = 5), the DB value measured for Bc differed from that for DTB(n = 5, P < 0.05). In addition, the calibrators made from Bc based different matrices, such as HSA, BSA and human serum, were significantly different in DB/Bc, but no difference was seen among the concentrations. Furthermore, the DB values determined for DTB or Bc increased linearly with the corresponding concentrations, respectively, with no difference between the slopes (YBc = 0.8300XBc + 1.9463, rBc2 = 0.9977, P < 0.01; YDTB = 0.8853XDTB-0.0251, rDTB2 = 0.9986, P < 0.01; n1 = n2 = 5, Pb1-b2 > 0.05).

The results demonstrate that the diazo reaction characters of Bc are identified with those of DTB. However, under the condition of DB, Bc reacts differently from DTB. This study also indicates that as a calibrator of DB based human serum, Bc has the similar constant effect of HCl as serum samples do, so it is a more reliable calibrator to eliminate the matrix effects.