title: A modification of the acid diazo coupling method (Malloy-Evelyn) for the determination of serum total bilirubin.
abstract: A simple and reliable method for the determination of total bilirubin from human serum is described. In this method, indirect bilirubin is liberated by the tenside in 0.12 mol l-1 HCl (R1), and the total bilirubin is coupled with a 2,5-dichlorobenzene diazonium (DBD) salt to obtain the corresponding azobilirubin having a lambda max of about 520-522 nm. The method can easily be applied to the KONE Delta, a fully automated, discrete random access clinical analyser, and also to less modern instruments. A sample volume of 5 microliters, R1 volume of 180 microliters, and R2 volume of 36 microliters was used on the KONE Delta. After a 5-min incubation 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 mumol l (n = 10). The between-run imprecision was from 2.2 to 1.3% within the range 13-97 mumol l-1 (n = 8). The method is linear up to at least 340 mumol l-1 (19.8 mg dl-1), and dilution extends the test limit to 3400 mumol l-1 (198.8 mg dl-1). The linearity of dilution was good over the practical 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 compartment lasts at least 2 weeks.
authors.affiliation: Department of Clinical Chemistry, University of Kuopio, Finland.
authors.forename: M T
authors.initials: MT
authors.lastname: Parviainen
mesh_terms: D001322:Autoanalysis / Q000379:methods / Q000592:standards; D001663:Bilirubin / Q000097:blood* / Q000592:standards; D004396:Coloring Agents; D003979:Diazonium Compounds; D006801:Humans; D012015:Reference Standards; D015203:Reproducibility of Results; D013053:Spectrophotometry / Q000592:standards; D013501:Surface-Active Agents
chemical_list: D004396:Coloring Agents; D003979:Diazonium Compounds; D013501:Surface-Active Agents; C013990:Fast Scarlet GG; D001663:Bilirubin