High-performance liquid chromatography coupled to enzyme-amplified biochemical detection for the analysis of hemoglobin after pre-column biotinylation

The determination of proteins with enzyme-amplified biochemical detection ( EA-BCD) coupled on-line with highperformance liquid chromatography (HPLC) i s demonstrated. The EA-BCD system was developed to detect biotin-containing compounds. Hemoglobin, which was used as a model compound, was biotinylate d prior to sample introduction. Several biotinylation parameters, such as p H and removal of excess biotinylation reagent, were investigated. After bio tinylation samples were introduced to HPLC followed by EA-BCD. To the HPLC effluent, alkaline phosphatase label streptavidin (S-AP) was added, which p ossesses high affinity to biotin and biotin-containing compounds. Excess S- AP was removed by means of an immobilized biotin column followed by substra te addition. The non-fluorescent substrate is converted to a highly fluores cent product by the enzyme label. A detection limit of 2 femtomol biotinyla ted Hb was achieved with good reproducibility and linearity. However, bioti nylation at low analyte concentration suffers from low yield due to slow re action kinetics. Finally, Hb was successfully extracted from urine with a r ecovery of 94%. (C) 2000 Elsevier Science B.V. All rights reserved.