Fluorimetric determination of dopamine in pharmaceutical products and urine using ethylene diamine as the fluorigenic reagent
Introduction
Dopamine, a neurotransmitter, is one of the naturally occurring catecholamines, and its hydrochloride salt is used in the treatment of acute congestive and renal failure [1]. Many analytical chemists try to find compendious methods for the determination of dopamine in authentic and dosage forms. Wang et al. studied the fluorescence property of dopamine and reported a fluorimetric determination of dopamine in injection and urine samples [2]. da Vieira and Fatibello-Fo published a spectrophotometric method for determining dopamine using a crude extract of sweet potato root as enzymatic source [3]. N-hydroxysuccinimidyl 3-indolylacetate [4] and 1,2-bis(3-chlorophenyl)ethylenediamine [5] had been employed as pre-column derivatization reagents for determining catecholamines by liquid chromatography (LC). Based on oxidation by N-bromosuccinimide [6] and the charge transfer reaction between dopamine and tetrachlorobenzoquinone [7], spectrophotometric methods for determining dopamine in pharmaceutical formulations have been developed. The effect of micelles on the electrochemistry of dopamine have also been studied, and dopamine was determined in the presence of a 100 times excess of ascorbic acid [8]. A sample mixture containing dopamine, catechol, adrenaline and noradrenaline was separated and determined by the electrochemical method [9]. Wang et al. published a fluorimetric method for determining dopamine in different parts of the brain stem and spinal cord [10]. The trihydroxyindole method [11] and an improved trihydroxyindole method using iodine as oxidation reagent and sodium sulfite as stabilization reagent [12] are widely used for determining dopamine. The trihydroxyindole method has good selectivity, but the stability and the detection limit are not satisfactory. Based on the reaction of catecholamines with 1,2-dipentylethylene diamine, catecholamines have been determined by fluorimetry, but the reaction time was longer [13]. A highly sensitive method based on a terbium ion fluorescence probe for the determination of dopamine has been reported [14].
In this paper, a new method for determining dopamine in injection and urine samples was developed. Dopamine was oxidized by mercury(II) nitrate and the oxidation product condensed with ethylene diamine to form a fluorescent substance. According to the literature [15], we presumed that the fluorescent substance was a quinoxaline derivative (I):
The measurement of the relative fluorescence intensity (RFI) of product I was carried out at 447 nm with excitation at 393 nm in a pH 4.0 HCl–NaOAc buffer solution. Under optimum condition, a linear relationship was obtained between the relative fluorescence intensity and the concentration of dopamine in the range 0.02–0.6 μg ml−1. Generally, the detection limit of spectrophotometry is 2–3 orders of magnitude higher than that of fluorimetry.
The principal advantage of the method described here is its low detection limit, which is four times lower than that of a previous fluorimetric method [2] and 10 times lower than that of another previous fluorimetric method [12]. This method can be used for the determination of dopamine in injection and urine samples. The results obtained by this method agreed with those obtained by the official method [16].
Section snippets
Apparatus
Spectrofluorimetric measurements were made on LS-5B (Perkin-Elmer) spectrofluorimeter equipped with a xenon discharge lamp and 1 cm quartz cells.
A liquid chromatograph (Model LC-6A, Shimadzu) was used for determining dopamine according to the official method [16].
A pH meter (Model pHS-3C, Shanghai Leici Instruments Factory, China) was used for monitoring pH adjustment.
Silica gel plates 0.2–0.25 mm thick, mm area (Qingdao Oceanic Departed Factory) were used to separate dopamine from urine
Excitation and emission spectra of product I
As can be seen (Fig. 1), the maximum excitation and emission wavelength of dopamine are at 393 and 447 nm, respectively (lines a and b). The Reagent blank has no effect on the determination of dopamine (lines c and d). Therefore, wavelengths of 393 and 447 nm were selected as excitation and emission wavelengths, respectively.
Selection of oxidant
The procedure is based on the dopamine reacted with oxidant to form an indole derivative, and the indole derivative condensed with ethylene diamine to form a quinoxaline
Conclusions
The results presented in this paper clearly demonstrate that dopamine can be determined by the fluorimetric method proposed. The results obtained agreed with those obtained by the LC method. The principal advantage of the proposed method is its low detection limit. This method can be used for determination of the dopamine in injection solutions of dopamine hydrochloride, and in urine samples.
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