A novel simultaneous injection effective mixing analysis system (SIEMA) for determination of direct bilirubin in urine sample was developed. Bilirubin reacts with diazotized sulfanilic acid in the presence of n-octyl-β-d-thioglucoside (OTG) as a solubilizing agent to form OTG-azobilirubin. The flow and chemical variables were investigated. A linear calibration graph for direct bilirubin was obtained over the range of 0-1.0 mg L(-1) (r(2)=0.994) with the limit of detection (3σ) of 4.7 μg L(-1), and the relative standard deviation (RSD) being 1.9% (n=11, 0.5 mg L(-1) of direct bilirubin). The results in healthy adult urine obtained by the proposed approach were found in good agreement with those obtained by the batch-wise diazo method.
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