Chemiluminescence determination of citrate and pyruvate and their mixtures by the stopped-flow mixing technique

A novel kinetic chemiluminescent method using the stopped-flow mixing technique has been investigated for the rapid and sensitive determination of citrate and pyruvate. The method is based on a tris(2,2′-bipyridiyl)ruthenium(III) (Ru(bpy)₃³⁺) chemiluminescence (CL) reaction. Ru(bpy)₃³⁺ was generated in the mixing chamber by oxidising tris(2,2′-bipyridyl)ruthenium(II) with cerium(IV). After selecting the best operating parameters, calibration graphs were obtained over the concentration ranges 0.38-38 μg ml⁻¹ and 8.7-1300 ng ml⁻¹ for citrate and pyruvate, respectively. The limits of detection were 0.1 μg ml⁻¹ for citrate and 0.3 ng ml⁻¹ for pyruvate. Based on the differential rate of the chemiluminescent reaction corresponding to citrate and pyruvate, a very simple kinetic procedure was developed for the simultaneous determination of both compounds. Mixtures of citrate and pyruvate in ratios between 15:1 and 1.5:1 were satisfactorily resolved. The proposed method was successfully applied to the determination of citrate in pharmaceutical formulations, pyruvate in animal blood serum and both compounds in human urine.