Alternative spectrophotometric method for determination of bilirubin and urobilinogen in urine samples using simultaneous injection effective mixing flow analysis

Abstract

A new approach based on simultaneous injection effective mixing flow analysis (SIEMA) system was developed for the successive spectrophotometric determination of urobilinogen and bilirubin in urinary samples. A 4-channel SIEMA system consisted of a syringe pump, two 5-cross connectors, four holding coils (two of four were used for urobilinogen determination, and other two of four were used for bilirubin determination), five solenoid valves, a mixing coil and a spectrophotometer. For urobilinogen assay, the color development was based on the reaction with p-diethylaminobenzaldehyde (p-DEABA) in the presence of strong hydrochloric acid. On the other hand, bilirubin was measured by the reaction with diazotized sulfanilic acid in the presence of n-octyl-β-D-thioglucoside (OTG) which served as a solubilizing agent to form OTG–azobilirubin. Firstly for bilirubin determination, aliquots of sample and reagent solutions were aspirated into two individual holding coils by a syringe pump, and then the aspirated zones were simultaneously dispensed in the reverse direction to the detector flow cell. Secondary for urobilinogen determination, aliquots of sample and reagent for urobilinogen were aspirated into other two holding coils, followed by the similar spectrophotometric detection. Under optimal conditions, the automated method provided linearity up to 100.0 mg L⁻¹ for urobilinogen and 5.0 mg L⁻¹ for bilirubin. The 3σ limit of detections (LODs) for urobilinogen and bilirubin were 1.0 mg L⁻¹ and 0.003 mg L⁻¹, respectively. The relative standard deviations (RSDs, n = 11) of 30 mg L⁻¹ urobilinogen and 1.0 mg L⁻¹ for bilirubin were 1.5% and 1.0%, respectively. Sample throughput of the whole assay of both urobilinogen and bilirubin was 30 h⁻¹. The proposed method can be applied to trace urobilinogen and bilirubin in urine samples and the results are useful for screening diabetic diagnostic.