A MODIFICATION OF THE ACID DIAZO COUPLING METHOD (MALLOY-EVELYN) FOR THE DETERMINATION OF SERUM TOTAL BILIRUBIN

A simple and reliable method for the determination of total bilirubin from human serum is described. In this method, indirect bilirubin is l iberated by the tenside in 0.12 mol l(-1) HCl (R1), and the total bili rubin is coupled with a 2,5-dichlorobenzene diazonium (DBD) salt to ob tain the corresponding azobilirubin having a lambda(max) of about 520- 522 nm. The method can easily be applied to the KONE Delta, a fully au tomated, discrete random access clinical analyser, and also to less mo dern instruments. A sample volume of 5 mu l, R1 volume of 180 mu l, an d R2 volume of 36 mu l was used on the KONE Delta. After a 5-min incub ation at 37 degrees C, measurement at 575 nm was done (main wavelength ). The within-run imprecision (CV%) varied from 2.9 to 0.3% within the serum total bilirubin range of 14-290 mu mol l (n = 10). The between- run imprecision was from 2.2 to 1.3% within the range 13-97 mu mol l(- 1) (n = 8). The method is linear up to at least 340 mu mol l(-1) (19.8 mg dl(-1)), and dilution extends the test limit to 3400 mu mol l(-1) (198.8 mg dl(-1)). The linearity of dilution was good over the practic al measuring range. The present method had a strong linear correlation with the Boehringer 2,5-dichlorophenyl diazonium (DPD) method on the Hitachi 717 analyser: y(DBD) = 1.018x(DPD) + 0.758, r = 0.9955 (n = 61 ). The stability of R2 (diazo reagent) in the analyser reagent compart ment lasts at least 2 weeks.