Spectrofluorimetric determination of heparin using a tetracycline-europium probe

A new spectrofluorimetric method was developed for the determination of trace amounts of heparin (Hep). Using tetracycline (TC)-europium ion (Eu3+) as a fluorescent probe, in the buffer solution at pH 8.8, Hep can remarkably enhance the fluorescence intensity of the TC-Eu3+ complex at lambda=612 nm and the enhanced fluorescence intensity of Eu3+ is in proportion to the concentration of Hep. Optimal conditions for the determination of Hep were also investigated. The linear range and detection limit for the determination of Hep were 0.02 to 1.6 microg/ml and 4.45 ng/ml, respectively. This method is simple, practical, and relatively free of interference from coexisting substances, and it can be applied successfully to assess Hep in biological samples. By the Rosenthal graphic method, the association constant and binding numbers of Hep with the probe were 4.46 x 10(4) L/mol and 16.2, respectively. Moreover, the enhancement mechanism of the fluorescence intensity in the TC-Eu3+ system, the TC-Eu(3+)-Hep system, and the TC-Eu(3+)-Hep-CTMAB system is also discussed.