A single nonaqueous diazo reagent for the rapid determination of bilirubin in serum without lipemic interference.

A nonaqueous reagent for directly determining serum bilirubin with 3,3'-dimethoxydiphenyl-4,4'-tetrazonium chloride in acidified dimethylsulfoxide is described. Conjugated or unconjugated bilirubin may be determined after extraction in acidified chloroform-ethyl acetate. The procedure is accurate (r2 0.97; recovery 100% for total, 98% for extracted bilirubin) and precise (C.V. 3.0% at 65 mg bilirubin per 1. The reagent develops endpoint color at 590 nm in less than 2 min, and the developed absorbance is stable for several hours. The method is intended for special applications where gross lipemia is encountered. Standardization with pure bilirubin in dimethylsulfoxide or chloroform is identical to standardization with aqueous bilirubin in protein standards.