Noradrenaline and dopamine are two of the most important monoamine neurotransmitters. They play important roles in learning and memory, and in regulation of cardiovascular activity and nervous system. Analysis of the monoamine neurotransmitters has conventionally used high performance liquid chromatography (HPLC) 1-3 or gas chromatography. 4,5 However, the determination of monoamine neurotransmitters in the biological matrix is very difficult because the compounds are usually present at very low concentrations, and many other compounds in biological matrix, which have also absorption in the low-wavelength region, interfere badly with the analysis of monoamine neurotransmitters. Cyanine derivative (Cy5) is a new fluorescence derivative reagent. The excitation and the emission wavelength of Cy5 are 649 and 670 nm, respectively. Thus diode lasers, which emit wavelengths from deep-red to infrared region, are chosen as the light source for laser-induced fluorescence (LIF). This is preferential for determining Cy5-labeled amine neurotransmitters, since almost all of the biological substances have no absorption band in the deep-red and infrared region, resulting in a remarkable decrease of background fluorescence noise and in an increase of detection sensitivity. Recently, LIF is widely used in the analysis of DNA sequence, oligonucleotides and proteins.
6-8But determination of Cy5-labeled monoamine neurotransmitters has not been reported. Capillary electrophoresis (CE) is a newly emerging technique for rapid and high-resolution separation of biomolecules.9 Recently, CE is widely used in the separation and determination of amine. 10 The detection methods of amine in CE include UV-VIS, 11 electrochemical detection 12 and LIF. 13 LIF is one of the most sensitive detection techniques in CE because of its low background signal and the small sample volume. In this paper, we report a rapid, sensitive method for the analysis of noradrenaline and dopamine in CE coupled diode LIF detection, in which Cy5 was chosen as the fluorescence derivative reagent.The effects of buffer concentration, pH value, and reaction time on the derivatization reaction of monoamines were investigated.
Experimental
ReagentsUnless stated otherwise, all chemicals were of analyticalreagent grade (Beijing Reagent Corp., Beijing, China). Noradrenaline, dopamine and sodium dodecyl sulfate (SDS) were purchased from Sigma (St. Louis, MO, USA). Cy5 was obtained from Amersham Pharmcia Biotech, USA. Water was deionized and purified by a Milli-Q Millipore purification system (Millipore, Marlborough, MA, USA).
ApparatusAll analyses were performed on a Beckman P/ACE 5000 system equipped with a Beckman Laser Module 635 nm. This was used as an exciting light source (Beckman-Fullerton, CA, USA). Separations were performed in fused-silica capillaries (57 cm × 75 µm i.d.; Yong Nian Optic Fiber Factory, Hebei Province, China).
Derivatization procedureNoradrenaline and dopamine were labeled with Cy5 as follows: Cy5 was dissolved in dimethylformamide. The solution with a...