Simultaneous quantification of catecholamines in rat brain by high-performance liquid chromatography with on-line...

A new method based on high-performance liquid chromatography (HPLC) coupled with on-line gold nanoparticle-catalyzed luminol chemiluminescence (CL) detection was developed for the simultaneous quantitation of catecholamines in rat brain. In the present CL system, gold nanoparticles were produced by the on-line reaction of H2 O2 , NaHCO3 -Na2 CO3 (buffer solution of luminol) and HAuCl4. Norepinephrine (NE), epinephrine (EP) and dopamine (DA) could strongly enhance the CL signal of the on-line gold nanoparticle-catalyzed luminol system. The UV-visible absorption spectra and transmission electron microscopy studies were carried out, and the CL enhancement mechanism was proposed. Catecholamines promoted the on-line formation of more gold nanoparticles, which better catalyzed the luminol-H2 O2 CL reaction. The good separation of NE, EP and DA was achieved with isocratic elution using a mixture of methanol and 0.2% aqueous phosphoric acid (5:95, v/v) within 8.5 min. Under the optimized conditions, the detection limits, defined as a signal-to-noise ratio of 3, were in the range of 1.32-1.90 ng/mL, corresponding to 26.4-38.0 pg for 20 μL sample injection. The recoveries of catecholamines added to rat brain sample were >94.6%, with the precisions <5.5%. The validated HPLC-CL method was successfully applied to determine NE and DA in rat brain without prior sample purification.