Simultaneous Determination of Dopamine and Carvedilol in Human Serum and Urine by First-order Derivative Fluorometry

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1 2005 The Japan Society for Analytical Chemistry 1281 Simultaneous Determination of Dopamine and Carvedilol in Human Serum and Urine by First-order Derivative Fluorometry Huai You WANG,* Yan X,IAO* Juan HAN,* and Xue Shun CHANG** *College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan , People s Republic of China **Third Occupation Technical School, Licheng Courty, Jinan , People s Republic of China A sensitive and selective method for simultaneous determination of carvedilol and dopamine was described. The emission wavelengths of carvedilol and dopamine were at 354 nm and 314 nm with the excitation at 290 nm, respectively. The determination of carvedilol and dopamine by normal fluorometry was difficult because the emission spectra of carvedilol and dopamine were overlapped seriously. The first derivative peaks of carvedilol and dopamine were at 336 nm and 302 nm, respectively. The linear regression equations of the calibration graphs of carvedilol and dopamine were C = H and C = H , with the correlation coefficients were and , respectively. The liner range for the determination of carvedilol was µg ml 1 to 0.02 µg ml 1, and 0.05 µg ml 1 to 0.6 µg ml 1 for dopamine. The detection limits were 1 ng ml 1 for carvedilol and 0.04 µg ml 1 for dopamine, respectively. The relative standard derivative (RSD) of 4.38% and 4.35% was observed for carvedilol and dopamine, respectively. The recovery of carvedilol was from 95.00% to 106.7% in human serum and from 97.50% to 105.0% in urine sample. The recovery of dopamine was from 100.0% to 102.5% in human serum and from 97.50% to 105.0% in urine sample. This method is simple and can be used for determination of carvedilol and dopamine in human serum and urine sample with satisfactory results. (Received January 13, 2005; Accepted March 11, 2005) Introduction Carvedilol is one of the medicines that can mitigate tardive movement disorders, psychosis and depression. 1,2 It is especially useful in treatment of heart failure. Dopamine, a neurotransmitter, can enhance the shrinking ability of cardiac muscle, increase the heart blood output, improve the vessel ending circulation and increase the urine volume. It can be used in the treatment of various types of shock, acute congestive failure and renal failure. 3 Based on their iatrical characters, carvedilol and dopamine are often used together. Therefore, it is important that a method of simultaneous determination of carvedilol and dopamine be developing. Some methods for determination of carvedilol by high-performance liquid chromatography and capillary electrophoresis in human serum, plasma, human tissue and rat plasma have been reported Wang et al. studied the fluorescence property of carvedilol and the contents of carvedilol in tablets by fluorometry, 11 simultaneous determination of carvedilol and ampicillin sodium by synchronous fluorometry was reported. 12 Flow-injection analytical methods, fluorescence methods, biosensor methods, electroanalysis methods using polymer-filmmodified electrode, nano-gold electrode 27 and molecularself-assembled-modified electrods, capillary electrophoresis 31,32 and high-performance liquid chromatography 33,34 have been reported for the determination of dopamine. To whom correspondence should be addressed. wanghuaiyou@sdnu.edu.cn In this paper, a first-order derivative fluorescence method was developed for the simultaneous determination of carvedilol and dopamine. The peak heights of first-order derivative spectra were estimated to be 302 nm (peak-to-zero) for dopamine and 336 nm (peak-to-valley) for carvedilol on the longitudinal coordinate axis (absolute value), respectively. Effects of ph, stability of carvedilol and dopamine and foreign ions on the determination of carvedilol and dopamine have been examined. This method was rapid, simple, and highly sensitive for the determination of carvedilol and dopamine without preseparation. This method can be used for the determination of carvedilol and dopamine in human serum and urine samples. Experiment Apparatus Spectrofluorimetric measurements were made on LS-50 (Perkin-Elmer, USA) spectrofluorimeter equipped with a xenon dischange lamp and 1 cm quartz cells. Derivative spectra can be evaluated using Fluorescence Data Manager (FLDM) software. A ph meter (Model phs-3c, Shanghai Leici Instruments Factory, China) was used for ph adjustment. Reagents and solutions Carvedilol and dopamine hydrochloride (>99.5%, chemical correspondent product), were used as standards (Chinese Drug and Biological Product Detection Institute). Other reagents were of analytical reagent grade. Doubly distilled water was used in all experiments.

2 1282 ANALYTICAL SCIENCES NOVEMBER 2005, VOL. 21 Stock standard solutions of carvedilol (1000 µg ml 1 ) and dopamine (1000 µg ml 1 ) An accurately weighed g standard sample of carvedilol was dissolved in 5 ml ethanol, transferred into 100 ml volumetric flask and diluted to the mark with water and mixed well. Dopamine hydrochloride g was dissolved in water directly; the mixture was transferred into a 100 ml volumetric flask and diluted to the mark with water and mixed well. The solutions were stored at 4 C. The carvedilol solution is stable at least one month and the dopamine hydrochloride solution is stable at least two weeks. The carvedilol solution was diluted to 1.00 µg ml 1 and the dopamine hydrochloride solution was diluted to 10.0 µg ml 1 before being used. Michaelis buffer solution (ph = 5.0) A total of 0.1 mol l 1 of HOAc and 0.1mol l 1 of NaOAc were mixed well according to the volume ratio of 1:2. Procedure Procedure I. A 1.0 ml portion of 0.10 µg ml 1 carvedilol was transferred into 10.0 ml volumetric flasks, 2.0 ml of ph 5.0 HOAc/NaOAc buffer solution was added; the mixture was diluted to the mark with water and mixed well. Dopamine standard solution was prepared with the same procedure. The fluorescence spectra of excitation and emission of the solutions were recorded. The scan rate of 240 nm min 1 and the excitation/ emission windows of 10 nm were used during all the experiment. Procedure II. A 1.0 ml portion of µg ml 1 carvedilol and 1.0 ml of 10.0 µg ml 1 dopamine standard solutions were transferred to 10.0 ml volumetric flask sequentially, 2.0 ml of ph 5.3 HOAc/NaOAc buffer solution was added; the contents were diluted to the mark with water and mixed well. The spectra of emission of the solutions were recorded with the excitation at 290 nm. The first-order derivative fluorescence spectra of the carvedilol and dopamine were obtained with FLDM software. A suitable number of points (30) was used for deriving first-order derivative spectra. The peak height of firstorder derivative spectrum was estimated at 302 nm (peak-tozero) for dopamine on the longitudinal coordinate axis (absolute value). Similarly, the peak height at 336 nm for carvedilol was estimated, too. Procedure III. Human blood samples containing carvedilol and dopamine from volunteers were collected and centrifuged for 3 min. Each supernatant was collected and stored at 20 C. One milliliter of the supernatant was transferred to 1000 ml volumetric flask and diluted to the mark with water and mixed well before being used. This solution was numbered sample solution 1. The sample solution 1 was used in determination of carvedilol and dopamine. Another human blood sample (not containing carvedilol or dopamine) was treated with the same way and numbered sample solution 2. Sample solution 2 was used for determination of recovery for carvedilol and dopamine in human serum. Procedure IV. Urine specimens containing carvedilol and dopamine from volunteers were collected in 10 ml of 6 mol l 1 hydrochloric acid over a 24 h period and stored at 4 C. One milliliter of this solution was transferred to 100 ml volumetric flask and diluted to the mark with water and mixed well before being used. This solution was numbered sample solution 3. The sample solution 3 was used as determination of carvedilol and dopamine. Other urine specimens (not containing carvedilol or dopamine) from volunteers was treated with the same procedure and numbered sample solution 4. The sample solution 4 was used for determination of recovery for carvedilol and dopamine in urine sample. Fig. 1 The fluorescence spectra of carvedilol and dopamine: Line a and line b are spectra of excitation and emission of carvedilol, respectively. Line c and line d are spectra of excitation and emission of dopamine. Concentration of carvedilol, 0.01 µg ml 1 ; concentration of dopamine, 1.0 µg ml 1. Fig. 2 The first-order derivative fluorescence spectra of the binary mixture: Concentration of carvedilol, 0.01 µg ml 1 ; concentration of dopamine, 1.0 µg ml 1. Results and Discussion The first-order derivative spectrum According to Procedure I, the excitation and emission spectra of carvedilol and dopamine were recorded and shown in Fig. 1. As can be seen (Fig. 1), the maximum excitation and emission wavelengths of carvedilol were at 250 nm and 354 nm, respectively. The maximum excitation and emission wavelengths of dopamine were at 290 nm and 314 nm, respectively. Obviously, the emission spectra of carvedilol and dopamine were overlapped seriously, although their maximum excitation wavelengths were different. As everyone knows, the shape of an emission spectrum is not related with excitation wavelength, 35 namely, the shape of emission spectrum does not change but the relative fluorescence intensity will change. How should one select a co-excitation wavelength for simultaneous determination of carvedilol and dopamine? Tests found that the detection limit of carvedilol was lower than that of dopamine, although 290 nm, the excitation wavelength of dopamine, was selected as the excitation wavelength of carvedilol (maximum excitation wavelength of carvedilol was 250nm); the relative fluorescence

3 1283 Table 1 Effect of foreign ions on the determination of carvedilol and dopamine Foreign ion Tolerance level/µg ml 1 K +, Na +, Cl, Ac, H 2PO 2 4, SO HPO Mg Ca 2+, NO Zn intensity of carvedilol was not down sharply with excitation of 290 nm. So 290 nm, the maximum excitation wavelength of dopamine, was selected as co-excitation wavelength for simultaneous determination of carvedilol and dopamine. Since the emission spectra of carvedilol and dopamine were overlapped, conventional fluorometry cannot be used to simultaneously determine carvedilol and dopamine. The derivative techniques can be applied to quantitative problems, especially for the analysis of mixtures of compounds whose spectra overlap. 36 Figure 2 shows that first-order derivative peaks of carvedilol and dopamine can be separated entirely, and thus carvedilol and dopamine can be simultaneously determined by the first-order derivative fluorometry. The first-order derivative peaks of carvedilol and dopamine were at 302 nm and 336 nm, respectively. Influence of ph Effects of ph on the relative fluorescence intensity of carvedilol and dopamine were tested; comparative tests at various ph values showed that the relative fluorescence intensities of carvedilol and dopamine changed with ph. The results are shown in Fig. 3. Variation of ph from 1.0 to 13.0 was investigated. It can be seen that relative fluorescence intensities of the both the drugs show no notable changes in the ph range from 1.0 to 7.0. Therefore, the ph = 5.0 HOAc/NaOAc buffer solution was selected to control ph of the system. In addition, the test showed that the relative fluorescence intensities of both drugs were stable for at least 6 h. Solvent effect According to Procedure II, the effects of organic solvents on the relative fluorescence intensities of carvedilol and dopamine were tested. The results showed that the relative fluorescence intensities of carvedilol and dopamine increased in the organic solvents tested, such as methanol, ethanol, isopropanol and N, N -dimethylformamide (DMF). In addition, the maximum emission wavelengths of carvedilol and dopamine caused red shifts in the presence of organic solvents and the shapes of firstorder derivative spectra changed, which resulted in the measurements of first-order derivative peak height becoming inaccurate. Furthermore, organic solvent should be forbidden in the sample treatment and determination. Effect of foreign ions A systematic study was carried out on the effects of foreign ions on the determination of carvedilol and dopamine. A 500 µg ml 1 level of each interfering species was tested. If the interference occurred, the ratio was decreased gradually till the interference ceased. The tolerance level was defined as an error not exceeding ± 5% in the determination of the analyte. The results are summarized in Table 1. Calibration graph of carvedilol and dopamine Under the selected conditions, a linear relationship was Fig. 3 The effect of ph on the fluorescence intensities of carvedilol (D) and dopamine (B). Concentration of carvedilol, 0.03 µg ml 1 ; dopamine, 10 µg ml 1. Fig. 4 The relationship of the height of the first-order derivative curve and the concentration of carvedilol. Fig. 5 The relationship of the height of the first-order derivative curve and the concentration of dopamine. obtained between the height of the first derivative curve and the concentration of carvedilol in the range of µg ml 1 to 0.02 µg ml 1. The linear regression equation of the calibration graph was C = H , with a correlation coefficient of linear regression of Samely, a linear range for dopamine was from 0.05 µg ml 1 to 0.6 µg ml 1 and the linear regression equation of calibration graph was C = H , with a correlation coefficient of linear regression of The results are shown in Figs. 4 and 5.

4 1284 ANALYTICAL SCIENCES NOVEMBER 2005, VOL. 21 Table 2 samples Results for the recovery of carvedilol in human serum ± ± ± ± ± ± 3.6 Table 4 Results for the recovery of carvedilol in urine samples ± ± ± ± ± ± 4.3 Table 3 samples Results for the recovery of dopamine in human serum ± ± ± ± ± ± 3.8 Table 5 Results for the recovery of dopamine in urine samples ± ± ± ± ± ± 3.3 Table 6 Results for the sample analysis Sample No. Human serum/mg ml 1 Urine/mg ml 1 Carvedilol Dopamine Carvedilol Dopamine ± ± ± ± ± ± ± ± ± ± ± ± Recoveries of carvedilol and dopamine in human serum sample According to Procedure II, the different concentrations of carvedilol and dopamine were added to sample solution 2 prepared according to Procedure III, and recoveries of carvedilol and dopamine were measured. The results are shown in Table 2 and Table 3, respectively. The confidence intervals were estimated for the results of determination in Table 4 and Table 5 based on P = 95% and n = 3. P is the confindence level and n means the times that the replicating determination was respeated. The recovery of carvedilol was from to 106.7% and the recovery of dopamine was from to 102.5% in human serum sample. Recoveries of carvedilol and dopamine in urine sample According to the Procedure II, the different concentrations of carvedilol and dopamine were added to sample solution 4 prepared according to Procedure IV, and recoveries of carvedilol and dopamine were measured. The results are shown in Table 4 and Table 5. The recovery of carvedilol was from to 105.0% and the recovery of dopamine was from to 105.0% in urine sample. Analysis of sample According to the Procedure II, the carvedilol and dopamine were measured in human serum sample and urine sample. The results were shown in Table 6. The human serum and urine samples were diluted to appropriate concentrations in order that carvedilol and dopamine concentrations locate in the range of calibration graph before carvedilol and dopamine in the samples were determined. Reproducibility and detection limit According to Procedure II, a portion of sample solution of carvedilol and dopamine was measured 10 times (n = 10). The mean value of carvedilol was µg ml 1 with a RSD of 4.38%, and the mean value of dopamine was 0.22 µg ml 1 with a RSD of 4.35%. Similarly, a reagent blank prepared with the same reagent concentration but no carvedilol and dopamine was measured 10 times (n = 10). The detection limits of carvedilol and dopamine were 1 ng ml 1 and 0.04 µg ml 1, respectively, based on the blank plus three times the standard deviation of the blank. 37 Conclusion This research has clearly demonstrated that carvedilol and dopamine can be simultaneously determined by derivative fluorimetry. This method is simple and can be used for determination of carvedilol and dopamine in human serum and urine. Acknowledgement We are grateful for the offers of human blood and urine samples from the Hospital of Shandong Normal University. References 1. W. H. Frishman, N. Engl. J. Med., 1998, 339, M. Pacher, M. R. Bristow, J. N. Cohn, W. S. Colucci, M. B. Fowler, E. M. Gilbert, and N. H. Shusterman, N. Engl. J. Med., 1996, 334, X.-Q. Chen and Y.-Y. Jin, New Pharmacology, 1998, Vol.14, People s Sanitation Press, Beijing, P. Ptábek, J. Macek, and J. Klíma, J. Chromatogr., B, 2003, 789, N. Hokama, N. Hobara, H. Kameya, S. Ohshiro, and M.

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