Fluorescent Determination of Noradrenaline and Dopamine Derivatized with Cy5 in Capillary Electrophoresis

Abstract

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...

Simultaneous Determination of Epinephrine, Noradrenaline and Dopamine in Human Serum Samples by High Performance Liquid Chromatography with Chemiluminescence Detection

“…Many methods have been proposed for the determination of CA, such as spectrophotometry, [2][3][4][5] thermal lens microscopy, 6 fluorimetry, [7][8][9] electrochemical method (ECD), [10][11][12][13][14] and capillary electrophoresis (CE). [15][16][17][18][19][20][21] In the past two decades, high performance liquid chromatography (HPLC) has become one of the most powerful and conceptually simple separation techniques for the analysis of CA because of its high resolution and short analysis time. At the same time, detection methods compatible with HPLC underwent continuous development including fluorescence detection (FL), [22][23][24][25] photodiode array detection (PDA) 26 and electrochemical detection (ECD).…”

Quantification of polyamines in human erythrocytes using a new near‐infrared cyanine 1‐(ϵ‐succinimidyl‐hexanoate)‐ 1′‐methyl‐3,3,3′,3′‐tetramethyl‐ indocarbocyanine‐5,5′‐disulfonate potassium with CE‐LIF detection

“…In earlier papers, Cy5 had been used for investigating the fluorescence labeling of proteins (BSA) [8][9][10][11] and diagnosing the 21-hydroxylase deficiency [12]. Amino acids [13,14], amantadine [15], mAb [16], monoamine neurotransmitters [17], and phosphorus-containing amino acid herbicides [18] have also been determined by Cy5 with diode LIF detection. To date, despite several methods have been established, few of them are involved in the quantification of biologically significant analytes derivatized by Cy5.…”

“…To date, despite several methods have been established, few of them are involved in the quantification of biologically significant analytes derivatized by Cy5. For example, only three reports have demonstrated the feasibility of quantitative determination of biologically significant amines using Cy5 by CE-LIF [15,17,18]. However, the proposed method for quantifying monoamine neurotransmitters [17] is not applied to the detection of monoamine neurotransmitters in real samples.…”

“…For example, only three reports have demonstrated the feasibility of quantitative determination of biologically significant amines using Cy5 by CE-LIF [15,17,18]. However, the proposed method for quantifying monoamine neurotransmitters [17] is not applied to the detection of monoamine neurotransmitters in real samples. Quantitative method for detecting amantadine in plasma suffers from low sensitivity and high interferences [15].…”

Rapid and sensitive determination of phosphorus-containing amino acid herbicides in soil samples by capillary zone electrophoresis with diode laser-induced fluorescence detection

“…An indocyanine fluorescence dye, sulfoindocyanine succinimidyl ester (Cy5), has recently been developed and used for investigating the fluorescence labeling of proteins (BSA) [20][21][22][23] and diagnosing the 21-hydroxylase deficiency [24]. Amino acids [25,26], amantadine [27], mAb [28], and monoamine neurotrasmitters such as noradrenaline and dopamine [29] have also been determined with high sensitivity by diode laser LIF combined with CZE [25,28] and MEKC [26,27,29].…”