FLOW-INJECTION SPECTROPHOTOMETRIC ENZYMATIC AND NONENZYMATIC METHODS FOR THE DETERMINATION OF DIRECT AND TOTAL BILIRUBIN IN SERUM

Two automatic spectrophotometric methods based on the use of flow-injection analysis are proposed for the determination of total and direct (conjugated) bilirubin in serum. The first method is based on the oxidation of bilirubin catalysed by bilirubin oxidase and it uses an adaptation of reagent injection and stopped flow modes for the sequential determination of total and direct bilirubin. The second method is based on the Erlich diazotation reaction and calls for the simultaneous determination of both bilirubin forms. The enzymatic method affords linear determination ranges between 0.09 and 25.0 and between 0.14 and 13.0 muM for total and direct bilirubin, respectively. The non-enzymatic method allows the determination of total and direct bilirubin with the linear ranges 0.85-144.0 and 3.80-125.0 muM, respectively. The relative standard deviation was 3% in all instances.