- Email: [email protected]
Fluorimetric determination of dopamine in pharmaceutical products and urine using ethylene diamine as the fluorigenic reagent
Analytica Chimica Acta 497 (2003) 93–99
Fluorimetric determination of dopamine in pharmaceutical products and urine using ethylene diamine as the fluorigenic reagent Huai You Wang a,∗ , Qiu Sha Hui b , Li Xiao Xu a , Ji Gang Jiang a , Yue Sun a a
College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China b Shandong Tradition Chinese Medicine University, Jinan 250014, China Received 5 March 2003; received in revised form 17 July 2003; accepted 26 August 2003
Abstract A sensitive and selective method for the determination of dopamine is described. Dopamine was oxidized by mercury(II) nitrate and the oxidation product was condensed with ethylene diamine to form a quinoxaline derivative which was strongly fluorescent. The measurement was carried out at 447 nm with excitation at 393 nm. Effects of pH, oxidants, and foreign ions on the determination of dopamine were examined. A linear relationship was obtained between the relative fluorescence intensity (RFI) and the concentration of dopamine in the range of 0.02 to 0.06 g ml−1 . The linear regression equation of the calibration graph is C = 0.001347F − 0.02564 (C is concentration of dopamine (g ml−1 ) and F is relative fluorescence intensity in the equation), with a correlation coefficient of 0.9991 and a relative standard deviation of 4.4%. Dopamine was separated from adrenaline and noradrenaline in urine by thin layer chromatography. The detection limit is 18 ng ml−1 , and the recovery is from 95.0 to 106.6%. This method can be used for the determination of dopamine in injection and urine samples. © 2003 Elsevier B.V. All rights reserved. Keywords: Dopamine; Ethylene diamine; Fluorimetry; Injection; Urine
1. 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 ∗ Corresponding author. Fax: +86-531-2615258. E-mail address: [email protected] (H.Y. Wang).
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
0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2003.08.050
94
H.Y. Wang et al. / Analytica Chimica Acta 497 (2003) 93–99
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 g 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].
2. Experimental 2.1. 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, 100 mm × 200 mm area (Qingdao Oceanic Departed Factory) were used to separate dopamine from urine samples.
2.2. Reagents and solutions Dopamine hydrochloride, adrenaline and noradrenaline (>99.8%, chemical correspondent product), were used as standards (Chinese Drug and Biological Product Detection Institute). All reagents were of analytical-reagent grade. Doubly distilled water was used in all experiments. A stock standard solution of dopamine hydrochloride (100 g ml−1 ) was prepared as follows: An accu-
H.Y. Wang et al. / Analytica Chimica Acta 497 (2003) 93–99
rately weighed 0.1000 g standard sample of dopamine hydrochloride was dissolved in water, transferred into a 100 ml volumetric flask, diluted to the mark with water and mixed well. The solution was stored at 4 ◦ C. The stock standard solution was diluted to 10.0 g ml−1 before use. Adrenaline (10.0 g ml−1 ) and noradrenaline (10.0 g ml−1 ) standard solutions were prepared according to the preparation procedure for dopamine. n-Butanols glacial acetic acid and water were mixed well in to volume ratio of 4:1:5. The solution was stood for half an hour, and the upper layer solution was used as the chromatographic developing reagent. HCl–NaOAc buffer solution (pH 4.0) was prepared as follows: 100 ml of 1 mol l−1 HCl and 80 ml of 1 mol l−1 NaOAc were transferred into a 250 ml standard flask, and diluted to the mark with water. Mercury(II) nitrate solution (0.1%, m/v) was prepared by adding 0.10 g of Hg(NO3 )2 to 1 ml of 0.01 mol l−1 HCl, diluting to 100 ml with water and mixing well. 2.3. Procedure I 0.4 ml portion of 10.0 g ml−1 dopamine standard solution was transferred into a 10 ml standard flask, 0.4 ml of Hg(NO3 )2 and 0.4 ml of ethylene diamine (1,2-diaminoethane) solutions were added. The mixed solution was shaken roughly, 2 ml of 0.4 mol l−1 HCl was added to neutralize excess ethylene diamine and 2 ml of HCl–NaOAc buffer solution was added. The mixture was heated for 10 min and then cooled in ice water at once, diluted to the mark with water and mixed well. The relative fluorescence intensity of the solution was measured at 447 nm with excitation at 393 nm against a reagent blank prepared with the same reagent concentrations but no dopamine hydrochloride. All fluorescence measurements were made using 5 nm excitation and emission windows at a scan rate of 240 nm min−1 .
95
The spots of dopamine, adrenaline and noradrenaline were colored. The Rf values were measured. 2.5. Procedure III Urine specimens from volunteers were collected in 10 ml of 6 mol l−1 hydrochloride acid over a 24-h period. 10 ml of such a urine sample was taken and evaporated to dryness in a vacuum at room temperature; the residue was dissolved in 2 ml of methanol. This solution was used as the sample solution. Fifteen microliters of sample solution was subjected to a silica gel plate by microdropper. The plate was developed in developing reagent for 3 h at room temperature, and dried in a desiccator. The spot of dopamine on the plate was scratched off (to ensure accuracy of sampling, the place of the spot of sample should be confirmed based on the standard dopamine), transferred into 10 ml centrifuge tube, 0.6 ml of buffer solution (HCl–NaOAc), 1.2 ml of methanol, 1 ml of sodium sulfite (0.05 mol l−1 ), and 0.2 ml water were added separately. The mixture was stirred for 10 min, stood for half an hour, and centrifuged for 2 min at 1500 rpm. The supernatant was analyzed according to procedure I.
3. Result and discussion 3.1. 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.
2.4. Procedure II
3.2. Selection of oxidant
Fifteen microliters of dopamine, adrenaline and noradrenaline standard solutions were subjected to a silica gel thin layer chromatography (TLC) plate by microdropper. The plate was developed in developing reagent for 3 h at room temperature, dried in a desiccator, and then merged into the vaporation of iodine.
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 derivative. The oxidant had an important role in the formation of product I. Many oxidants, such as silver oxide, lead dioxide, manganese
96
H.Y. Wang et al. / Analytica Chimica Acta 497 (2003) 93–99
Fig. 1. Excitation and emission spectra of product I and blank a and b are spectra of excitation and emission of product I, respectively, c and d are spectra of excitation and emission of blank, respectively.
dioxide, sodium peroxodisulphate, and cerium(IV) sulfate [17], were used as the oxidant for determining catecholamines. According to procedure I, oxidants, such as mercury(II) nitrate, sodium nitrate, potassium hexacyanoferrate(IV), hydrogen peroxide, cerium(IV) sulfate and oxygen were tested. The result shown that the blank value was too high when hydrogen peroxide or potassium hexacyanoferrate(III) was used as oxidant; and the relative fluorescence intensity of product I decreased more or less when cerium(IV) sulfate or O2 were used as the oxidant. The reaction speed was lower when sodium nitrate was used under the selected condition. The relative fluorescence intensity of product I was strongest when mercury nitrate was used as oxidant, so mercury nitrate was selected as oxidant. The result shown 0.4 ml of mercury nitrate solution (0.4%) was sufficient for determining dopamine up to 0.6 g ml−1 (upper limit of linear range). Therefore, 0.4 ml of mercury nitrate solution was recommended.
can be seen that the spectrum of product I was different at different pH values. The maximum emission wavelength was at 487 nm, at pH 1–2 and changed to 447 nm in the pH range 3–7; the maximum emission wavelength was at 452 nm in the pH range 8–11. It is
3.3. Influence of pH Effect of pH on the relative fluorescence intensity of product I has been tested; comparative tests at various pH values showed that the relative fluorescence intensity of product I changes with pH when using procedure I. The results are shown in Fig. 2. Variation of the pH from 1.0 to 11.0 was investigated. It
Fig. 2. Effect of pH on the emission spectra of product I. Lines 1–11 indicate pH 1.0–11.0, sequentially.
H.Y. Wang et al. / Analytica Chimica Acta 497 (2003) 93–99
likely that the different spectra arise from protonated and unprotonated form of product I. Because the relative fluorescence intensity of product I was maximal at pH 4.0, and there was an excellent linear relationship between fluorescence intensity and dopamine concentration at this pH, pH 4.0 was selected for use. Various buffer solutions of pH 4.00, HCl–NaOAc, potassium hydrogenphthalate/sodium hydroxide, and sodium citrate/hydrochloric acid, were tested. The results showed that HCl–NaOAc was the only buffer that did not interfere in the determination of dopamine, and therefore was selected to control the pH of the system.
97
Table 1 Effect of foreign ions on the determination of 0.4 g ml−1 dopamine Foreign ions or species
Tolerance level (g ml−1 )
Foreign ions or species
Tolerance level (g ml−1 )
Na+ , Cl− , K+ , NO3 − Mg2+ , Zn2+ , Pb2+ SO4 2− PO4 3− Glycine l-Phenylalanine dl-Tryptophan Noradrenaline
500 100 200 300 250 150 50 0.15
Cu2+ CO3 2− HPO4 2− Al3+ l-Lysine l-Tyrosine Adrenaline
5 10 40 5 40 50 0.20
3.4. Formation and stability of product I 3.7. Calibration graph for dopamine 0.4 g ml−1
By using procedure I, a solution of dopamine was heated for different times in a boiling water bath and the relative fluorescence intensity was measured. This showed that the formation of product I was too slow at room temperature, but when heated for 8–15 min in a boiling water bath, the relative fluorescence intensity became constant. Thus heating for 10 min was selected as optimum. In addition, the relative fluorescence intensity of product I was measured after standing for different times. After the 10-min heating period. This showed that the relative fluorescence intensity of product I was stable for at least 4 h. 3.5. Effect of foreign ions A systematic study was carried out on the effects of commonly found ions on the determination of 0.4 g ml−1 dopamine. A 500 mg ml−1 level of each potentially interfering ion was tested first. If interference occurred, the ratio was reduced progressively until interference ceased. The tolerance level was defined as an error not exceeding ±5% in the determination of the analyte. The results were summarized in the Table 1. 3.6. Effect of amount of ethylene diamine It was found that addition of 0.4 ml of ethylene diamine solution (1 mol l−1 ) was sufficient for determining dopamine up to 0.6 g ml−1 (upper limit of linear range). Therefore, 0.4 ml of ethylene diamine solution was recommended.
Results obtained under the selected conditions were plotted to give a calibration graph of relative fluorescence intensity versus concentration of dopamine. The results are shown in Table 2. According to this data, there is a linear relationship between the relative fluorescence intensity and the concentration of dopamine in the range 0.02–0.6 g ml−1 . The linear regression equation of the calibration graph is C = 0.001347F − 0.02564. The linear correlation coefficient of 0.9991 (n = 6). 3.8. Comparison of fluorimetric and liquid chromatographic methods Injection solutions of dopamine hydrochloride (different batch number) were diluted to different concentrations with water. These solutions analyzed as sample solutions, and the relative fluorescence intensities of the sample solutions were measured. The concentrations of dopamine in the samples were calculated from the linear calibration graph. Dopamine Table 2 The relationship between relative fluorescence intensity (RFI) and concentration of dopamine (C) C (g ml−1 )
RFI
0.1 0.2 0.3 0.4 0.5 0.6
91.0 166 252 310 390 465
98
H.Y. Wang et al. / Analytica Chimica Acta 497 (2003) 93–99
Table 3 Determination of dopamine in injection (mean ± 95% confidence, n = 3) Sample number
Fluorimetry (mg ml−1 )
1 2 3 4
0.10 0.50 0.75 2.11
± ± ± ±
0.01 0.02 0.01 0.03
LC (mg ml−1 ) 0.09 0.51 0.73 2.09
± ± ± ±
0.02 0.01 0.02 0.02
in these sample solutions was also measured by liquid chromatography according to the British Pharmacopeia [16]. The results are shown in Table 3. As can be seen the results obtained by fluorimetry agreed with those of LC. In addition, the four groups of data for the two methods in Table 3 were tested with the Cochran method [18]. At a confidence level (P) equal to 95%, there was no significant difference between the two methods. 3.9. Recovery of dopamine The recovery of dopamine added to different concentrations of sample is shown in Table 4. The recovery ranged from 95.0 to 106.6%. 3.9.1. Determination of Rf values of dopamine, adrenaline and noradrenaline Dopamine, adrenaline and noradrenaline are similar in molecular structure, and the latter two compounds will interfere in the determination of dopamine. Thus TLC was investigated to separate the compounds. It was found that n-butanol–glacial acetic acid–water as developing reagent was optimum for separation of dopamine. By use of procedure II, the Rf values of the compounds were measured, and for dopamine, adrenaline and noradrenaline were found to be 0.64, 0.44, and 0.92, respectively. Dopamine was separated completely from adrenaline and noradrenaline.
Table 5 Determination of dopamine in urine (mean ± 95% confidence, n = 3) Sample number
Fluorimetry (mg ml−1 )
LC (mg ml−1 )
1 2 3
0.051 ± 0.005 0.020 ± 0.002 0.065 ± 0.001
0.053 ± 0.002 0.018 ± 0.003 0.063 ± 0.002
3.9.2. Analysis of urine sample By use of procedure III, the dopamine in a urine sample was measured. The results are shown in Table 5, which agree with those obtained by the LC. 3.9.3. Reproducibility and detection limit A portion of the sample solution of dopamine was transferred into a 10 ml standard flask, the reagent was added and the fluorescence developed and measured according to procedure I. This sample solution was measured 10 times, the mean value was 0.498 g ml−1 with a relative standard deviation of 4.38%. When a reagent blank, treated in the same way was measured 10 times, the detection limit was found to be 18 ng ml−1 , based on the blank plus three times the standard deviation of the blank [19].
4. 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. References
Table 4 Recovery of dopamine from injection solution of dopamine (confidence intervals from Table 3) Sample number
Sample content (g ml−1 )
Dopamine (g ml−1 )
1 2 3
0.100 ± 0.010 0.300 ± 0.015 0.350 ± 0.012
0.100 0.150 0.200
Added Found
Recovery (%)
0.196 ± 0.001 96.0 ± 1.0 0.460 ± 0.003 106.6 ± 2.6 0.540 ± 0.005 95.0 ± 2.5
[1] B.K. George, in: L.S. Goodman, A. Gilman (Eds.), The pharmacological Basis of Therapeutics, 3rd ed., The Macmillan Company, New York, 1965, p. 427. [2] H.Y. Wang, Y. Sun, B. Tang, Talanta 57 (2002) 899. [3] I.C. da Vieira, O. Fatibello-Fo, Talanta 46 (1998) 559. [4] H. Wang, H. Jin, H.S. Zhang, Fresenius J. Anal. Chem. 365 (1999) 682. [5] G.H. Ragab, H. Nohta, K. Zaitsu, Anal. Chim. Acta 403 (2000) 155.
H.Y. Wang et al. / Analytica Chimica Acta 497 (2003) 93–99 [6] P. Nagaraja, K.C. Srnivasa Murthy, K.S. Rangappa, N.M. Made Gowda, Talanta 46 (1998) 39. [7] Y. Long, D.H. Li, J.Z. Feng, S.Y. Tong, Fenxi Huaxue 25 (1997) 916. [8] X.L. Wen, Y.H. Jia, Z.L. Liu, Talanta 50 (1999) 1027. [9] L. Hua, S.N. Tan, Anal. Chim. Acta 403 (2000) 179. [10] C.Y. Wang, Z.G. Pang, B.Q. Wang, P. Zhang, Fenxi huaxue 27 (1999) 101. [11] W. Schmollack, A. Steup, H. Bekemeier, Pharmazie 39 (1984) 30. [12] K. Oka, M. Sekiya, H. Osada, K. Fujita, T. Kato, T. Nagatsu, Clin. Chem. 28 (1982) 646. [13] H. Nohta, A. Mitsui, Y. Ohkara, Anal. Chim. Acta 165 (1984) 171.
99
[14] X. Wu, S.L. Tong, B.Y. Su, F. Huang, J.H. Yang, Fenxi Huaxue 27 (1999) 1069. [15] T. Siki, M. Hamaji, J. Chromatogr. 162 (1980) 388. [16] The British Pharmacopoeia, British Pharmacopoeia Commission, vol. 1, HMSO, London, 1998, p. 1911. [17] M.E. El-kommos, F.A. Mohamed, A.S. Khder, Talanta 37 (1990) 625. [18] Y.X. Zheng, The Method of Mathematical Statistics in Analytical Chemistry, Science Press, Beijing, 1986, pp. 122, 231, 308. [19] ACS Committee on Environmental improvement, Anal. Chem. 52 (1980) 2242.
Fluorimetric determination of dopamine in pharmaceutical products and urine using ethylene diamine as the fluorigenic reagent Huai You Wang a,∗ , Qiu Sha Hui b , Li Xiao Xu a , Ji Gang Jiang a , Yue Sun a a
College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China b Shandong Tradition Chinese Medicine University, Jinan 250014, China Received 5 March 2003; received in revised form 17 July 2003; accepted 26 August 2003
Abstract A sensitive and selective method for the determination of dopamine is described. Dopamine was oxidized by mercury(II) nitrate and the oxidation product was condensed with ethylene diamine to form a quinoxaline derivative which was strongly fluorescent. The measurement was carried out at 447 nm with excitation at 393 nm. Effects of pH, oxidants, and foreign ions on the determination of dopamine were examined. A linear relationship was obtained between the relative fluorescence intensity (RFI) and the concentration of dopamine in the range of 0.02 to 0.06 g ml−1 . The linear regression equation of the calibration graph is C = 0.001347F − 0.02564 (C is concentration of dopamine (g ml−1 ) and F is relative fluorescence intensity in the equation), with a correlation coefficient of 0.9991 and a relative standard deviation of 4.4%. Dopamine was separated from adrenaline and noradrenaline in urine by thin layer chromatography. The detection limit is 18 ng ml−1 , and the recovery is from 95.0 to 106.6%. This method can be used for the determination of dopamine in injection and urine samples. © 2003 Elsevier B.V. All rights reserved. Keywords: Dopamine; Ethylene diamine; Fluorimetry; Injection; Urine
1. 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 ∗ Corresponding author. Fax: +86-531-2615258. E-mail address: [email protected] (H.Y. Wang).
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
0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2003.08.050
94
H.Y. Wang et al. / Analytica Chimica Acta 497 (2003) 93–99
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 g 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].
2. Experimental 2.1. 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, 100 mm × 200 mm area (Qingdao Oceanic Departed Factory) were used to separate dopamine from urine samples.
2.2. Reagents and solutions Dopamine hydrochloride, adrenaline and noradrenaline (>99.8%, chemical correspondent product), were used as standards (Chinese Drug and Biological Product Detection Institute). All reagents were of analytical-reagent grade. Doubly distilled water was used in all experiments. A stock standard solution of dopamine hydrochloride (100 g ml−1 ) was prepared as follows: An accu-
H.Y. Wang et al. / Analytica Chimica Acta 497 (2003) 93–99
rately weighed 0.1000 g standard sample of dopamine hydrochloride was dissolved in water, transferred into a 100 ml volumetric flask, diluted to the mark with water and mixed well. The solution was stored at 4 ◦ C. The stock standard solution was diluted to 10.0 g ml−1 before use. Adrenaline (10.0 g ml−1 ) and noradrenaline (10.0 g ml−1 ) standard solutions were prepared according to the preparation procedure for dopamine. n-Butanols glacial acetic acid and water were mixed well in to volume ratio of 4:1:5. The solution was stood for half an hour, and the upper layer solution was used as the chromatographic developing reagent. HCl–NaOAc buffer solution (pH 4.0) was prepared as follows: 100 ml of 1 mol l−1 HCl and 80 ml of 1 mol l−1 NaOAc were transferred into a 250 ml standard flask, and diluted to the mark with water. Mercury(II) nitrate solution (0.1%, m/v) was prepared by adding 0.10 g of Hg(NO3 )2 to 1 ml of 0.01 mol l−1 HCl, diluting to 100 ml with water and mixing well. 2.3. Procedure I 0.4 ml portion of 10.0 g ml−1 dopamine standard solution was transferred into a 10 ml standard flask, 0.4 ml of Hg(NO3 )2 and 0.4 ml of ethylene diamine (1,2-diaminoethane) solutions were added. The mixed solution was shaken roughly, 2 ml of 0.4 mol l−1 HCl was added to neutralize excess ethylene diamine and 2 ml of HCl–NaOAc buffer solution was added. The mixture was heated for 10 min and then cooled in ice water at once, diluted to the mark with water and mixed well. The relative fluorescence intensity of the solution was measured at 447 nm with excitation at 393 nm against a reagent blank prepared with the same reagent concentrations but no dopamine hydrochloride. All fluorescence measurements were made using 5 nm excitation and emission windows at a scan rate of 240 nm min−1 .
95
The spots of dopamine, adrenaline and noradrenaline were colored. The Rf values were measured. 2.5. Procedure III Urine specimens from volunteers were collected in 10 ml of 6 mol l−1 hydrochloride acid over a 24-h period. 10 ml of such a urine sample was taken and evaporated to dryness in a vacuum at room temperature; the residue was dissolved in 2 ml of methanol. This solution was used as the sample solution. Fifteen microliters of sample solution was subjected to a silica gel plate by microdropper. The plate was developed in developing reagent for 3 h at room temperature, and dried in a desiccator. The spot of dopamine on the plate was scratched off (to ensure accuracy of sampling, the place of the spot of sample should be confirmed based on the standard dopamine), transferred into 10 ml centrifuge tube, 0.6 ml of buffer solution (HCl–NaOAc), 1.2 ml of methanol, 1 ml of sodium sulfite (0.05 mol l−1 ), and 0.2 ml water were added separately. The mixture was stirred for 10 min, stood for half an hour, and centrifuged for 2 min at 1500 rpm. The supernatant was analyzed according to procedure I.
3. Result and discussion 3.1. 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.
2.4. Procedure II
3.2. Selection of oxidant
Fifteen microliters of dopamine, adrenaline and noradrenaline standard solutions were subjected to a silica gel thin layer chromatography (TLC) plate by microdropper. The plate was developed in developing reagent for 3 h at room temperature, dried in a desiccator, and then merged into the vaporation of iodine.
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 derivative. The oxidant had an important role in the formation of product I. Many oxidants, such as silver oxide, lead dioxide, manganese
96
H.Y. Wang et al. / Analytica Chimica Acta 497 (2003) 93–99
Fig. 1. Excitation and emission spectra of product I and blank a and b are spectra of excitation and emission of product I, respectively, c and d are spectra of excitation and emission of blank, respectively.
dioxide, sodium peroxodisulphate, and cerium(IV) sulfate [17], were used as the oxidant for determining catecholamines. According to procedure I, oxidants, such as mercury(II) nitrate, sodium nitrate, potassium hexacyanoferrate(IV), hydrogen peroxide, cerium(IV) sulfate and oxygen were tested. The result shown that the blank value was too high when hydrogen peroxide or potassium hexacyanoferrate(III) was used as oxidant; and the relative fluorescence intensity of product I decreased more or less when cerium(IV) sulfate or O2 were used as the oxidant. The reaction speed was lower when sodium nitrate was used under the selected condition. The relative fluorescence intensity of product I was strongest when mercury nitrate was used as oxidant, so mercury nitrate was selected as oxidant. The result shown 0.4 ml of mercury nitrate solution (0.4%) was sufficient for determining dopamine up to 0.6 g ml−1 (upper limit of linear range). Therefore, 0.4 ml of mercury nitrate solution was recommended.
can be seen that the spectrum of product I was different at different pH values. The maximum emission wavelength was at 487 nm, at pH 1–2 and changed to 447 nm in the pH range 3–7; the maximum emission wavelength was at 452 nm in the pH range 8–11. It is
3.3. Influence of pH Effect of pH on the relative fluorescence intensity of product I has been tested; comparative tests at various pH values showed that the relative fluorescence intensity of product I changes with pH when using procedure I. The results are shown in Fig. 2. Variation of the pH from 1.0 to 11.0 was investigated. It
Fig. 2. Effect of pH on the emission spectra of product I. Lines 1–11 indicate pH 1.0–11.0, sequentially.
H.Y. Wang et al. / Analytica Chimica Acta 497 (2003) 93–99
likely that the different spectra arise from protonated and unprotonated form of product I. Because the relative fluorescence intensity of product I was maximal at pH 4.0, and there was an excellent linear relationship between fluorescence intensity and dopamine concentration at this pH, pH 4.0 was selected for use. Various buffer solutions of pH 4.00, HCl–NaOAc, potassium hydrogenphthalate/sodium hydroxide, and sodium citrate/hydrochloric acid, were tested. The results showed that HCl–NaOAc was the only buffer that did not interfere in the determination of dopamine, and therefore was selected to control the pH of the system.
97
Table 1 Effect of foreign ions on the determination of 0.4 g ml−1 dopamine Foreign ions or species
Tolerance level (g ml−1 )
Foreign ions or species
Tolerance level (g ml−1 )
Na+ , Cl− , K+ , NO3 − Mg2+ , Zn2+ , Pb2+ SO4 2− PO4 3− Glycine l-Phenylalanine dl-Tryptophan Noradrenaline
500 100 200 300 250 150 50 0.15
Cu2+ CO3 2− HPO4 2− Al3+ l-Lysine l-Tyrosine Adrenaline
5 10 40 5 40 50 0.20
3.4. Formation and stability of product I 3.7. Calibration graph for dopamine 0.4 g ml−1
By using procedure I, a solution of dopamine was heated for different times in a boiling water bath and the relative fluorescence intensity was measured. This showed that the formation of product I was too slow at room temperature, but when heated for 8–15 min in a boiling water bath, the relative fluorescence intensity became constant. Thus heating for 10 min was selected as optimum. In addition, the relative fluorescence intensity of product I was measured after standing for different times. After the 10-min heating period. This showed that the relative fluorescence intensity of product I was stable for at least 4 h. 3.5. Effect of foreign ions A systematic study was carried out on the effects of commonly found ions on the determination of 0.4 g ml−1 dopamine. A 500 mg ml−1 level of each potentially interfering ion was tested first. If interference occurred, the ratio was reduced progressively until interference ceased. The tolerance level was defined as an error not exceeding ±5% in the determination of the analyte. The results were summarized in the Table 1. 3.6. Effect of amount of ethylene diamine It was found that addition of 0.4 ml of ethylene diamine solution (1 mol l−1 ) was sufficient for determining dopamine up to 0.6 g ml−1 (upper limit of linear range). Therefore, 0.4 ml of ethylene diamine solution was recommended.
Results obtained under the selected conditions were plotted to give a calibration graph of relative fluorescence intensity versus concentration of dopamine. The results are shown in Table 2. According to this data, there is a linear relationship between the relative fluorescence intensity and the concentration of dopamine in the range 0.02–0.6 g ml−1 . The linear regression equation of the calibration graph is C = 0.001347F − 0.02564. The linear correlation coefficient of 0.9991 (n = 6). 3.8. Comparison of fluorimetric and liquid chromatographic methods Injection solutions of dopamine hydrochloride (different batch number) were diluted to different concentrations with water. These solutions analyzed as sample solutions, and the relative fluorescence intensities of the sample solutions were measured. The concentrations of dopamine in the samples were calculated from the linear calibration graph. Dopamine Table 2 The relationship between relative fluorescence intensity (RFI) and concentration of dopamine (C) C (g ml−1 )
RFI
0.1 0.2 0.3 0.4 0.5 0.6
91.0 166 252 310 390 465
98
H.Y. Wang et al. / Analytica Chimica Acta 497 (2003) 93–99
Table 3 Determination of dopamine in injection (mean ± 95% confidence, n = 3) Sample number
Fluorimetry (mg ml−1 )
1 2 3 4
0.10 0.50 0.75 2.11
± ± ± ±
0.01 0.02 0.01 0.03
LC (mg ml−1 ) 0.09 0.51 0.73 2.09
± ± ± ±
0.02 0.01 0.02 0.02
in these sample solutions was also measured by liquid chromatography according to the British Pharmacopeia [16]. The results are shown in Table 3. As can be seen the results obtained by fluorimetry agreed with those of LC. In addition, the four groups of data for the two methods in Table 3 were tested with the Cochran method [18]. At a confidence level (P) equal to 95%, there was no significant difference between the two methods. 3.9. Recovery of dopamine The recovery of dopamine added to different concentrations of sample is shown in Table 4. The recovery ranged from 95.0 to 106.6%. 3.9.1. Determination of Rf values of dopamine, adrenaline and noradrenaline Dopamine, adrenaline and noradrenaline are similar in molecular structure, and the latter two compounds will interfere in the determination of dopamine. Thus TLC was investigated to separate the compounds. It was found that n-butanol–glacial acetic acid–water as developing reagent was optimum for separation of dopamine. By use of procedure II, the Rf values of the compounds were measured, and for dopamine, adrenaline and noradrenaline were found to be 0.64, 0.44, and 0.92, respectively. Dopamine was separated completely from adrenaline and noradrenaline.
Table 5 Determination of dopamine in urine (mean ± 95% confidence, n = 3) Sample number
Fluorimetry (mg ml−1 )
LC (mg ml−1 )
1 2 3
0.051 ± 0.005 0.020 ± 0.002 0.065 ± 0.001
0.053 ± 0.002 0.018 ± 0.003 0.063 ± 0.002
3.9.2. Analysis of urine sample By use of procedure III, the dopamine in a urine sample was measured. The results are shown in Table 5, which agree with those obtained by the LC. 3.9.3. Reproducibility and detection limit A portion of the sample solution of dopamine was transferred into a 10 ml standard flask, the reagent was added and the fluorescence developed and measured according to procedure I. This sample solution was measured 10 times, the mean value was 0.498 g ml−1 with a relative standard deviation of 4.38%. When a reagent blank, treated in the same way was measured 10 times, the detection limit was found to be 18 ng ml−1 , based on the blank plus three times the standard deviation of the blank [19].
4. 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. References
Table 4 Recovery of dopamine from injection solution of dopamine (confidence intervals from Table 3) Sample number
Sample content (g ml−1 )
Dopamine (g ml−1 )
1 2 3
0.100 ± 0.010 0.300 ± 0.015 0.350 ± 0.012
0.100 0.150 0.200
Added Found
Recovery (%)
0.196 ± 0.001 96.0 ± 1.0 0.460 ± 0.003 106.6 ± 2.6 0.540 ± 0.005 95.0 ± 2.5
[1] B.K. George, in: L.S. Goodman, A. Gilman (Eds.), The pharmacological Basis of Therapeutics, 3rd ed., The Macmillan Company, New York, 1965, p. 427. [2] H.Y. Wang, Y. Sun, B. Tang, Talanta 57 (2002) 899. [3] I.C. da Vieira, O. Fatibello-Fo, Talanta 46 (1998) 559. [4] H. Wang, H. Jin, H.S. Zhang, Fresenius J. Anal. Chem. 365 (1999) 682. [5] G.H. Ragab, H. Nohta, K. Zaitsu, Anal. Chim. Acta 403 (2000) 155.
H.Y. Wang et al. / Analytica Chimica Acta 497 (2003) 93–99 [6] P. Nagaraja, K.C. Srnivasa Murthy, K.S. Rangappa, N.M. Made Gowda, Talanta 46 (1998) 39. [7] Y. Long, D.H. Li, J.Z. Feng, S.Y. Tong, Fenxi Huaxue 25 (1997) 916. [8] X.L. Wen, Y.H. Jia, Z.L. Liu, Talanta 50 (1999) 1027. [9] L. Hua, S.N. Tan, Anal. Chim. Acta 403 (2000) 179. [10] C.Y. Wang, Z.G. Pang, B.Q. Wang, P. Zhang, Fenxi huaxue 27 (1999) 101. [11] W. Schmollack, A. Steup, H. Bekemeier, Pharmazie 39 (1984) 30. [12] K. Oka, M. Sekiya, H. Osada, K. Fujita, T. Kato, T. Nagatsu, Clin. Chem. 28 (1982) 646. [13] H. Nohta, A. Mitsui, Y. Ohkara, Anal. Chim. Acta 165 (1984) 171.
99
[14] X. Wu, S.L. Tong, B.Y. Su, F. Huang, J.H. Yang, Fenxi Huaxue 27 (1999) 1069. [15] T. Siki, M. Hamaji, J. Chromatogr. 162 (1980) 388. [16] The British Pharmacopoeia, British Pharmacopoeia Commission, vol. 1, HMSO, London, 1998, p. 1911. [17] M.E. El-kommos, F.A. Mohamed, A.S. Khder, Talanta 37 (1990) 625. [18] Y.X. Zheng, The Method of Mathematical Statistics in Analytical Chemistry, Science Press, Beijing, 1986, pp. 122, 231, 308. [19] ACS Committee on Environmental improvement, Anal. Chem. 52 (1980) 2242.