Performance of the Continuous Addition of Reagent Technique in Fluorimetric Reaction-Rate Methods. Determination of Thiamine at the Nanomolar Level

Abstract

The potential of the continuous addition of reagent (CAR) technique, developed for reactions on the millisecond time scale, in fluorimetric reaction-rate methods, is evaluated. The approach was applied to the classical fast reaction involving the oxidation of thiamine to fluorescent thiochrome effected by potassium ferricyanide in basic solutions. Practical problems associated with this system were readily avoided by using the CAR technique. Several kinetic methods have been developed for the determination of thiamine; all are very sentitive (detection limit, 2. 5x10⁻⁹ M) compared to other fluorimetric equilibrium and reaction-rate methods and are suitable for routine analyses (sample throughput, 100 h⁻¹).

Key Words:

• Continuous addition
• fluorimetry
• reaction-rate method
• thiamine