Determination of total bilirubin in human serum by chemiluminescence from the reaction of bilirubin and peroxynitrite

Peroxynitrous acid (ONOOH) was produced by the on-line reaction of acidified hydrogen peroxide with nitrite in a flow system, and peroxynitrite (ONOO^-) was generated from ONOOH in NaOH solution. A weak chemiluminescent (CL) emission was observed due to the production of singlet oxygen (¹O₂) during the decomposition of ONOO^-. Bilirubin and its conjugate were found to enhance the CL emission of ¹O₂ in a suitable micellar medium. For the first time, the feasibility of employing the present CL system for the sensitive and selective determination of total bilirubin contents in human serum was demonstrated and the results were compared with certified values. The present method showed a great improvement on overcoming bis(2,4,6-trichlorophenyl)oxalate CL highly insolubility in aqueous solution and exhibiting higher tolerance towards interferences than redox reaction of bilirubin with various oxidizing agents such as sodium hypochlorite and iodine. The recoveries of bilirubin were found to fall in the range between 95 and 108%. The detection limits (S/N=3) for bilirubin and its conjugate were determined to be 10 and 8 ng ml^-1, respectively. The relative standard deviations (R.S.D.) for the consecutive CL detection of a series of bilirubin (30 μg l^-1) and bilirubin ditaurite (25 μg l^-1) were 3.2 and 2.9% (n=11), respectively.