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Flow injection chemiluminescence determination of catecholamines with electrogenerated hypochlorite
Analytica Chimica Acta 374 (1998) 105±110
Flow injection chemiluminescence determination of catecholamines with electrogenerated hypochlorite Chengxiao Zhanga, Jiachu Huang1,a, Zhujun Zhanga,*, Masuo Aizawab a
b
Department of Chemistry, Shaanxi Normal University, Xian 710062, China Department of Bioengineering, Tokyo Institute of Technology, Nagatsta, Midori-ku, Yokohama 226, Japan Received 18 February 1998; received in revised form 11 June 1998; accepted 20 June 1998
Abstract A ¯ow injection method for the determination of catecholamines based on the inhibition of the intensity of chemiluminescence from the luminol±hypochlorite system is described. The hypochlorite was electrogenerated on-line by constant current electrolysis, resulting in the elimination of the instability of hypochlorite solution prepared from commercially available sodium hypochlorite. The detection limits are 610ÿ10 g mlÿ1 for dopamine, 810ÿ10 g mlÿ1 for adrenaline and 810ÿ10 g mlÿ1 for isoprenaline. Linear ranges are 110ÿ9±210ÿ8 g mlÿ1 for dopamine, 210ÿ9± 210ÿ8 g mlÿ1 for adrenaline, 210ÿ9±210ÿ8 g mlÿ1 for isoprenaline. The relative standard deviations are lower than 2.8%. The proposed method has been applied to the determination of catecholamines in pharmaceutical injections with satisfactory results. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Flow injection; Chemiluminescence; Catecholamine; Luminol; Electrolysis; Pharmaceutical analysis
1. Introduction Catecholamine is now widely used in the treatment of bronchial asthma, hypertension, heart failure associated with organic heart disease, and in cardiac surgery [1]. The determination of catecholamines in pharmaceutical preparations and biological ¯uids has appeared especially attractive. A variety of techniques have been utilized to the determination of catecholamines, such as chromatography [1], spectrophoto*Corresponding author. Fax: +86-29-5308748; e-mail: [email protected] 1 Permanent address: Lu'an Teacher's College, Lu'an, Anhui 237012, China. 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00419-X
metry [2], spectro¯uorimetry [3], electrochemical detection [4], chemiluminescence [5,6] and capillary electrophoresis with luminescence detection [7]. Some of these methods lack sensitivity and speci®city. Chemiluminescence (CL) has been exploited in a number of analytical applications owing to its great sensitivity [8]. The usage of CL±¯ow injection analysis (FIA) as a simple mean of drug detection has been applied to many drugs including morphine [9], buprenorphine hydrochloride [10], promethazine [11], kanamycin [12], anaesthetics [13], and bilirubin [14]. In the CL reaction, mostly H2O2, O2, I2, KMnO4, KIO4, ClOÿ are used as oxidation reagents. Some CL reactions have hardly found any applications due to the instability of oxidation reagents such as hypo-
106
C. Zhang et al. / Analytica Chimica Acta 374 (1998) 105±110
chlorite [15,16], and superoxide. We proposed that unstable reagents required for the CL reaction can be generated electrochemically on-line in order to extend the application of CL analysis. We found that drugs containing polyphenol group can inhibit the CL emission from the luminol±hypochlorite system. In this paper, a simple, sensitive and rapid new assay for catecholamines (dopamine, adrenaline and isoprenaline) has been developed using an electrogenerated hypochlorite and ¯ow-injection CL technique. The proposed method has been applied to the determination of catecholamines in pharmaceutical injections with satisfactory results. 2. Experimental 2.1. Reagents All the reagents were of analytical grade and the water used was deionized and doubly distilled. Dopamine, adrenaline, isoprenaline are purchased from Shanghai Harvest Pharmaceutical. Stock standard solutions (1.0 mg mlÿ1 in water) were stored in the refrigerator. Working standard solutions were prepared daily from the stock solution by appropriate dilution. A 1.010ÿ2 M of stock luminol solution was prepared by dissolving 0.1172 g of luminol in 100 ml of 0.10 M sodium hydroxide, 0.1 M potassium chloride±0.01 M phosphate buffer solution (PBS) (pH 10.0). 2.2. Apparatus The CL±FIA system used in this work is shown in Fig. 1. A peristaltic pump was used to deliver all ¯ow streams at a ¯ow rate of 2 ml minÿ1 on each line. PTFE tubing (0.8 mm i.d.) was used to connect all components in the ¯ow system. Injection was made using a HPLC injector equipped with a 160 ml injector loop. The streams of luminol, sodium hydroxide, hypochlorite electrogenerated from an electrochemical cell and analyte were combined in a ¯ow cell. The signal produced in the ¯ow cell was detected with a R 456 photomultiplier tube and recorded with an XWT204 recorder (Shanghai Dahua Instrument and Meter Plant). Hypochlorite was generated on-line by the galvanostatic oxidation of potassium chloride solution
Fig. 1. Schematic diagram of the CL±FIA system for catecholamines: (a) luminol solution; (b) 0.10 M sodium hydroxide solution; (c) sample solution; (d) potassium chloride solution; P peristaltic pump; V injection valve; E electrochemical cell; G galvanostat; M mixing tube; F flow cell; PMT photomultiplier tube; A amplifier; R recorder; NHV negative high voltage.
using a two-compartment H-type glass cell in which cathodic and anodic chambers were separated from each other by a glass frit. A spiral platinum wire ( 0.2 mm100 mm) placed in ¯ow line and a spiral platinum wire (area 1.5 cm2) were used as the working and counter electrodes, respectively. The constant galvanostatic electrolysis was performed using a galvanostat (Shanghai Electric Instrument Plant). 2.3. Procedure As shown in Fig. 1, ¯ow lines were inserted into the luminol solution, sodium hydroxide solution, potassium chloride solution and sample/standard solution, respectively. Electrolysis was started to generate hypochlorite. Then 160 ml of luminol solution was injected in the sodium hydroxide solution. This stream was merged with a stream of sample and hypochlorite in ¯ow cell, producing CL emission. The concentration of sample was quanti®ed by the decreased CL intensity II0ÿIS, where IS is the CL intensity of the sample, and I0 is the blank signal. 3. Results and discussion 3.1. Choice of manifold and FI technique The determination of catecholamines is based on the inhibition of the luminol±hypochlorite CL system. The polyphenol group occurs in catecholamines such as dopamine, adrenaline and isoprenaline, and preliminary experiments showed that these compounds
C. Zhang et al. / Analytica Chimica Acta 374 (1998) 105±110
Fig. 2. Typical chemiluminescence kinetic response curves for determination of catecholamines in the absence (a) and presence (b) of 1.010ÿ8 g mlÿ1 dopamine.
exhibited the same reaction characteristics. Dopamine was chosen as a representative analyte in the optimization of experimental parameters. The chemiluminescence kinetic characteristics of the reaction of luminol and hypochlorite in basic solution were studied in detail. Fig. 2 shows the typical peak-shape responses for the detection of dopamine in the FIA system. It was found that the rate of the reaction in solution was very fast: from the reagent mixing to the peak maximum only 5 s were needed and it took 15 s for the signal to return to baseline. In the presence of dopamine, only peakheight (CL intensity) reduced. Several manifold arrangements and FIA systems were tested using similar conditions. Injection of analyte or oxidant instead of luminol was found to lead to poor reproducibility. In a system where the luminol solution was injected into a sample stream and mixed with the hypochlorite stream and then with a continuously ¯owing sodium hydroxide stream, the decrease in CL intensity obtained was not very large (ca. 15 mV). In a similar system in which the luminol solution was injected into the hydroxide solution stream instead of the sample stream solution, the intensity obtained was about 25% larger. In a third arrangement, luminol solution was injected into a
107
hydroxide stream as a carrier stream. Then in front of the detector this stream was mixed with the hypochlorite solution, which has been mixed with analyte in a ®xed mixing tube, so that a steady light emission was obtained and an inhibition signal directly proportional to the concentration of the analyte could be recorded. Catecholamines, which are strongly reducing reagents, can reduce hypochlorite so that they can decrease the CL intensity generated from the hypochlorite±luminol system. However, other strong reagents such as ascorbic acid, phenol decreased the CL intensity much lower than catecholamines. Therefore, it is not only a way that catecholamines reduce hypochloride to decrease the CL intensity. Preliminary experiments showed that mixing of the reaction solution of hypochlorite with catecholamines can strongly decrease the CL intensity. It is possible that the reaction products of hypochlorite with catecholamines lead to inhibition or catecholamines may interact with luminol in a way that leads to inhibition. The mechanism of the chemiluminescent reaction is under investigation. 3.2. Effect of length of mixing tube To ensure the ef®cient reaction between hypochlorite and analyte, a mixing tube was used in this system and its length was tested from 5 to 20 cm. It was found that a suitable length with a high decreased CL intensity was 12 cm, shorter or longer than this length would reduce the decrease in CL intensity because of a de®cient reaction or considerable hypochlorite decomposition. 3.3. Effect of electrolyte pH Fig. 3 illustrates the results of the electrolyte pH optimization. The decreased CL intensity increased rapidly from pH 8.0 to 9.5 and the maximum decreased CL intensity was obtained at pH 10.0. Above pH 11.0, the decreased CL intensity reduced probably because of de®cient generation of hypochlorite at the platinum electrode or the tendency of the hypochlorite ion is disproportionate in strong basic solution. Thus, a pH of 10.0 was chosen as optimum for the electrolyte stream and used for further experiments.
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C. Zhang et al. / Analytica Chimica Acta 374 (1998) 105±110
3.0 mA because the concentration of electrogenerated hypochlorite increases. This suggests that the concentration of electrogenerated hypochlorite can be controlled. For electrolysis currents higher than 3.0 mM, the decreased CL intensity remained almost constant, probably due to the excess of hypochlorite. The concentration of hypochlorite electrogenerated with a current of 4.0 mA was evaluated on the basis of Faraday's Law to be about 6.210ÿ4 M. The electrolysis current of 4.0 mA was selected as optimum. 3.5. Effect of potassium chloride concentration
Fig. 3. Effect of electrolyte pH on the decreased CL intensity for 1.010ÿ8 g mlÿ1 dopamine, 1.010ÿ5 M luminol, 0.10 M potassium chloride, electrolysis current: 0.40 mA.
3.4. Effect of electrolysis current The dependence of the decreased CL intensity on the electrolysis current was investigated using a stream consisting of a solution of 0.10 M KCl± 0.010 M PBS (pH 10.0). Fig. 4 shows the results in the sample stream containing 1.010ÿ8 g mlÿ1 analyte. The decreased CL signal increases from 0 to
Fig. 4. Effect of electrolysis current on the decreased CL intensity for 1.010ÿ8 g mlÿ1 dopamine, 1.010ÿ5 M luminol, 0.10 M phosphate buffer containing 0.10 M potassium chloride (pH 10.0).
Potassium chloride is used as the precursor participating in the reaction needed for the CL reaction and in the supporting electrolyte. The concentration of the potassium chloride in the line is expected to affect the concentration of hypochlorite electrogenerated. The in¯uence of the potassium chloride concentration in the line on decreased CL intensity is shown in Fig. 5. In the absence of potassium chloride in the PBS stream, no blank CL emission was observed. This suggests that dissolved oxygen originating from the oxidization of water cannot oxidize luminol producing the light. The decreased CL intensity became larger with increasing concentration of potassium chloride. Above the concentration of 0.080 M, it remained
Fig. 5. Effect of potassium chloride concentration on the decreased CL intensity for 1.010ÿ8 g mlÿ1 dopamine, 1.010ÿ5 M luminol, electrolysis current: 0.40 mA.
C. Zhang et al. / Analytica Chimica Acta 374 (1998) 105±110
109
obtained for dopamine, adrenaline and isoprenaline. The linear dynamic ranges, the correlation, detection limits and the relative standard deviations are summarized in Table 1. At a ¯ow rate of 2.0 ml minÿ1, the determination of analyte could be performed in 1 min including sampling and washing, giving a throughput of about 60 times per hour. 3.8. Application
Fig. 6. Effect of luminol concentration on the decreased CL intensity for 1.010ÿ8 g mlÿ1 dopamine, 0.10 M phosphate buffer containing 0.10 M potassium chloride (pH 10.0), electrolysis current: 0.40 mA.
almost constant. Thus the potassium chloride concentration of 0.10 M was chosen as optimum. 3.6. Effect of luminol concentration The in¯uence of the luminol concentration on the analytical signal was investigated. Fig. 6 shows the variation of the decreased CL intensity with the concentration of luminol solution. The greatest signal intensity was obtained when 1.010ÿ5 M luminol was used. In order to ensure maximum sensitivity, a luminol concentration of 1.010ÿ5 M was chosen in this work. 3.7. Calibration data Under the optimized condition and by use of the manifold depicted in Fig. 1, calibration curves were
In order to assess the possible application of the proposed method to the analysis of pharmaceutical dosage forms, the in¯uence of commonly used excipients and additives was investigated for the determination of 1.010ÿ8 g mlÿ1 catecholamines. No interference could be observed when the sample includes up to a 20-fold weight concentrations of fructose, glucose, lactose, sucrose, glycine, alanine, gum acacia, cellulose, or starch in the original catecholamines-containing powder. Additionally, the test also showed that more than 1000-fold excess of Ca2, Mg2, acetic acid, tartaric acid, citric acid, and less than 50-fold excess of uric acid, cholesterol, ascorbic acid did not interfere with the determination of 1.010ÿ8 g mlÿ1 catecholamines. Usually only one catecholamine is present in pharmaceutical preparations. The method may be suitable for adrenaline and isoprenaline determination in medicine. For biological ¯uids its applicability should be assessed carefully. The proposed method was applied to the analysis of some commercial injections. The results are shown in Table 2. As can be seen, there are no signi®cant differences between the labelled values and those obtained by the proposed method. In conclusion, the unstable reagents required for the CL reaction can be easily electrogenerated on-line and lead to methods for many potential applications. The proposed method for the determination of catechol-
Table 1 Parameters of the calibration curves used for the determination of dopamine, adrenaline and isoprenaline Catecholamine
Linear dynamic ranges (g mlÿ1)
Correlation coefficient (n5)
Detection limits (g mlÿ1)
RSD (%) (n11)
Dopamine Adrenaline Isoprenaline
110ÿ9±210ÿ8 210ÿ9±210ÿ8 210ÿ9±210ÿ8
0.9982 0.9975 0.9972
610ÿ10 110ÿ9 110ÿ9
2.4 2.8 2.6
110
C. Zhang et al. / Analytica Chimica Acta 374 (1998) 105±110
Table 2 Determination of three catecholamines in pharmaceutical preparations Preparations
Labelled value (g mlÿ1)
Present methoda found (g mlÿ1)
Recoverya (%)
RSDa (%)
Dopamine injection Adrenaline injection Isoprenaline injection
1.0 0.5 0.5
1.02 0.49 0.51
102 98 103
3.4 3.7 3.9
a
Average of three determinations.
amines by CL±FIA also demonstrates that CL has many potential applications in the area of pharmaceutical analysis. The method is superior to other conventional methods in that it is very fast, simple, and sensitive. Further work on this subject is in progress, aiming at liquid chromatographic applications. Acknowledgements This study was supported by the National Natural Science Foundation of China (no. 39730160). References [1] A.J. Pesce, L.A. Kaplan, S. Bircher (Ed.), Methods in Clinical Chemistry, C.V. Mosby, St. Louis, MO, 1987, p. 944. [2] J.J. Berzas Nevado, J.M. Lemus Gallego, P. Buitrago Laguna, Anal. Chim. Acta 300 (1995) 293.
[3] A. Kojlo, J. Martinez Calatayud, Anal. Chim. Acta 308 (1995) 334. [4] S. Uchiyama, S. Suzuki, Anal. Chim. Acta 261 (1992) 361. [5] J. Li, J. Lu, Chinese J. Anal. Chem. 25 (1997) 314. [6] A.A. Al-Warthan, S.A. Al-Tamarah, A.A. Al-Akel, Anal. Sci. 10 (1994) 449. [7] R. Zhu, W.Th. Kok, Anal. Chem. 69 (1997) 4010. [8] A. Townshend, Analyst 115 (1990) 495. [9] R.W. Abbott, A. Townshend, R. Gill, Analyst 111 (1986) 635. [10] A.A. Alwarthan, A. Townshend, Anal. Chim. Acta 185 (1986) 329. [11] A.A. Alwarthan, S.A. Al-Tamrah, A.A. Akel, Anal. Chim. Acta 282 (1993) 169. [12] A.A. Alwarthan, S.A. Al-Tamrah, A.A. Akel, Anal. Chim. Acta 292 (1994) 201. [13] X.R. Zhang, W.R.G. Baeyens, G. Van der Weken, A.C. Calokerinos, K. Imai, Anal. Chim. Acta 303 (1995) 137. [14] L.P. Palilis, A.C. Calokerinos, N. Grekas, Anal. Chim. Acta 333 (1996) 267. [15] W.R. Seitz, J. Phys. Chem. 79 (1975) 101. [16] D.F. Marino, J.D. Ingle Jr., Anal. Chem. 53 (1981) 445.
Flow injection chemiluminescence determination of catecholamines with electrogenerated hypochlorite Chengxiao Zhanga, Jiachu Huang1,a, Zhujun Zhanga,*, Masuo Aizawab a
b
Department of Chemistry, Shaanxi Normal University, Xian 710062, China Department of Bioengineering, Tokyo Institute of Technology, Nagatsta, Midori-ku, Yokohama 226, Japan Received 18 February 1998; received in revised form 11 June 1998; accepted 20 June 1998
Abstract A ¯ow injection method for the determination of catecholamines based on the inhibition of the intensity of chemiluminescence from the luminol±hypochlorite system is described. The hypochlorite was electrogenerated on-line by constant current electrolysis, resulting in the elimination of the instability of hypochlorite solution prepared from commercially available sodium hypochlorite. The detection limits are 610ÿ10 g mlÿ1 for dopamine, 810ÿ10 g mlÿ1 for adrenaline and 810ÿ10 g mlÿ1 for isoprenaline. Linear ranges are 110ÿ9±210ÿ8 g mlÿ1 for dopamine, 210ÿ9± 210ÿ8 g mlÿ1 for adrenaline, 210ÿ9±210ÿ8 g mlÿ1 for isoprenaline. The relative standard deviations are lower than 2.8%. The proposed method has been applied to the determination of catecholamines in pharmaceutical injections with satisfactory results. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Flow injection; Chemiluminescence; Catecholamine; Luminol; Electrolysis; Pharmaceutical analysis
1. Introduction Catecholamine is now widely used in the treatment of bronchial asthma, hypertension, heart failure associated with organic heart disease, and in cardiac surgery [1]. The determination of catecholamines in pharmaceutical preparations and biological ¯uids has appeared especially attractive. A variety of techniques have been utilized to the determination of catecholamines, such as chromatography [1], spectrophoto*Corresponding author. Fax: +86-29-5308748; e-mail: [email protected] 1 Permanent address: Lu'an Teacher's College, Lu'an, Anhui 237012, China. 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00419-X
metry [2], spectro¯uorimetry [3], electrochemical detection [4], chemiluminescence [5,6] and capillary electrophoresis with luminescence detection [7]. Some of these methods lack sensitivity and speci®city. Chemiluminescence (CL) has been exploited in a number of analytical applications owing to its great sensitivity [8]. The usage of CL±¯ow injection analysis (FIA) as a simple mean of drug detection has been applied to many drugs including morphine [9], buprenorphine hydrochloride [10], promethazine [11], kanamycin [12], anaesthetics [13], and bilirubin [14]. In the CL reaction, mostly H2O2, O2, I2, KMnO4, KIO4, ClOÿ are used as oxidation reagents. Some CL reactions have hardly found any applications due to the instability of oxidation reagents such as hypo-
106
C. Zhang et al. / Analytica Chimica Acta 374 (1998) 105±110
chlorite [15,16], and superoxide. We proposed that unstable reagents required for the CL reaction can be generated electrochemically on-line in order to extend the application of CL analysis. We found that drugs containing polyphenol group can inhibit the CL emission from the luminol±hypochlorite system. In this paper, a simple, sensitive and rapid new assay for catecholamines (dopamine, adrenaline and isoprenaline) has been developed using an electrogenerated hypochlorite and ¯ow-injection CL technique. The proposed method has been applied to the determination of catecholamines in pharmaceutical injections with satisfactory results. 2. Experimental 2.1. Reagents All the reagents were of analytical grade and the water used was deionized and doubly distilled. Dopamine, adrenaline, isoprenaline are purchased from Shanghai Harvest Pharmaceutical. Stock standard solutions (1.0 mg mlÿ1 in water) were stored in the refrigerator. Working standard solutions were prepared daily from the stock solution by appropriate dilution. A 1.010ÿ2 M of stock luminol solution was prepared by dissolving 0.1172 g of luminol in 100 ml of 0.10 M sodium hydroxide, 0.1 M potassium chloride±0.01 M phosphate buffer solution (PBS) (pH 10.0). 2.2. Apparatus The CL±FIA system used in this work is shown in Fig. 1. A peristaltic pump was used to deliver all ¯ow streams at a ¯ow rate of 2 ml minÿ1 on each line. PTFE tubing (0.8 mm i.d.) was used to connect all components in the ¯ow system. Injection was made using a HPLC injector equipped with a 160 ml injector loop. The streams of luminol, sodium hydroxide, hypochlorite electrogenerated from an electrochemical cell and analyte were combined in a ¯ow cell. The signal produced in the ¯ow cell was detected with a R 456 photomultiplier tube and recorded with an XWT204 recorder (Shanghai Dahua Instrument and Meter Plant). Hypochlorite was generated on-line by the galvanostatic oxidation of potassium chloride solution
Fig. 1. Schematic diagram of the CL±FIA system for catecholamines: (a) luminol solution; (b) 0.10 M sodium hydroxide solution; (c) sample solution; (d) potassium chloride solution; P peristaltic pump; V injection valve; E electrochemical cell; G galvanostat; M mixing tube; F flow cell; PMT photomultiplier tube; A amplifier; R recorder; NHV negative high voltage.
using a two-compartment H-type glass cell in which cathodic and anodic chambers were separated from each other by a glass frit. A spiral platinum wire ( 0.2 mm100 mm) placed in ¯ow line and a spiral platinum wire (area 1.5 cm2) were used as the working and counter electrodes, respectively. The constant galvanostatic electrolysis was performed using a galvanostat (Shanghai Electric Instrument Plant). 2.3. Procedure As shown in Fig. 1, ¯ow lines were inserted into the luminol solution, sodium hydroxide solution, potassium chloride solution and sample/standard solution, respectively. Electrolysis was started to generate hypochlorite. Then 160 ml of luminol solution was injected in the sodium hydroxide solution. This stream was merged with a stream of sample and hypochlorite in ¯ow cell, producing CL emission. The concentration of sample was quanti®ed by the decreased CL intensity II0ÿIS, where IS is the CL intensity of the sample, and I0 is the blank signal. 3. Results and discussion 3.1. Choice of manifold and FI technique The determination of catecholamines is based on the inhibition of the luminol±hypochlorite CL system. The polyphenol group occurs in catecholamines such as dopamine, adrenaline and isoprenaline, and preliminary experiments showed that these compounds
C. Zhang et al. / Analytica Chimica Acta 374 (1998) 105±110
Fig. 2. Typical chemiluminescence kinetic response curves for determination of catecholamines in the absence (a) and presence (b) of 1.010ÿ8 g mlÿ1 dopamine.
exhibited the same reaction characteristics. Dopamine was chosen as a representative analyte in the optimization of experimental parameters. The chemiluminescence kinetic characteristics of the reaction of luminol and hypochlorite in basic solution were studied in detail. Fig. 2 shows the typical peak-shape responses for the detection of dopamine in the FIA system. It was found that the rate of the reaction in solution was very fast: from the reagent mixing to the peak maximum only 5 s were needed and it took 15 s for the signal to return to baseline. In the presence of dopamine, only peakheight (CL intensity) reduced. Several manifold arrangements and FIA systems were tested using similar conditions. Injection of analyte or oxidant instead of luminol was found to lead to poor reproducibility. In a system where the luminol solution was injected into a sample stream and mixed with the hypochlorite stream and then with a continuously ¯owing sodium hydroxide stream, the decrease in CL intensity obtained was not very large (ca. 15 mV). In a similar system in which the luminol solution was injected into the hydroxide solution stream instead of the sample stream solution, the intensity obtained was about 25% larger. In a third arrangement, luminol solution was injected into a
107
hydroxide stream as a carrier stream. Then in front of the detector this stream was mixed with the hypochlorite solution, which has been mixed with analyte in a ®xed mixing tube, so that a steady light emission was obtained and an inhibition signal directly proportional to the concentration of the analyte could be recorded. Catecholamines, which are strongly reducing reagents, can reduce hypochlorite so that they can decrease the CL intensity generated from the hypochlorite±luminol system. However, other strong reagents such as ascorbic acid, phenol decreased the CL intensity much lower than catecholamines. Therefore, it is not only a way that catecholamines reduce hypochloride to decrease the CL intensity. Preliminary experiments showed that mixing of the reaction solution of hypochlorite with catecholamines can strongly decrease the CL intensity. It is possible that the reaction products of hypochlorite with catecholamines lead to inhibition or catecholamines may interact with luminol in a way that leads to inhibition. The mechanism of the chemiluminescent reaction is under investigation. 3.2. Effect of length of mixing tube To ensure the ef®cient reaction between hypochlorite and analyte, a mixing tube was used in this system and its length was tested from 5 to 20 cm. It was found that a suitable length with a high decreased CL intensity was 12 cm, shorter or longer than this length would reduce the decrease in CL intensity because of a de®cient reaction or considerable hypochlorite decomposition. 3.3. Effect of electrolyte pH Fig. 3 illustrates the results of the electrolyte pH optimization. The decreased CL intensity increased rapidly from pH 8.0 to 9.5 and the maximum decreased CL intensity was obtained at pH 10.0. Above pH 11.0, the decreased CL intensity reduced probably because of de®cient generation of hypochlorite at the platinum electrode or the tendency of the hypochlorite ion is disproportionate in strong basic solution. Thus, a pH of 10.0 was chosen as optimum for the electrolyte stream and used for further experiments.
108
C. Zhang et al. / Analytica Chimica Acta 374 (1998) 105±110
3.0 mA because the concentration of electrogenerated hypochlorite increases. This suggests that the concentration of electrogenerated hypochlorite can be controlled. For electrolysis currents higher than 3.0 mM, the decreased CL intensity remained almost constant, probably due to the excess of hypochlorite. The concentration of hypochlorite electrogenerated with a current of 4.0 mA was evaluated on the basis of Faraday's Law to be about 6.210ÿ4 M. The electrolysis current of 4.0 mA was selected as optimum. 3.5. Effect of potassium chloride concentration
Fig. 3. Effect of electrolyte pH on the decreased CL intensity for 1.010ÿ8 g mlÿ1 dopamine, 1.010ÿ5 M luminol, 0.10 M potassium chloride, electrolysis current: 0.40 mA.
3.4. Effect of electrolysis current The dependence of the decreased CL intensity on the electrolysis current was investigated using a stream consisting of a solution of 0.10 M KCl± 0.010 M PBS (pH 10.0). Fig. 4 shows the results in the sample stream containing 1.010ÿ8 g mlÿ1 analyte. The decreased CL signal increases from 0 to
Fig. 4. Effect of electrolysis current on the decreased CL intensity for 1.010ÿ8 g mlÿ1 dopamine, 1.010ÿ5 M luminol, 0.10 M phosphate buffer containing 0.10 M potassium chloride (pH 10.0).
Potassium chloride is used as the precursor participating in the reaction needed for the CL reaction and in the supporting electrolyte. The concentration of the potassium chloride in the line is expected to affect the concentration of hypochlorite electrogenerated. The in¯uence of the potassium chloride concentration in the line on decreased CL intensity is shown in Fig. 5. In the absence of potassium chloride in the PBS stream, no blank CL emission was observed. This suggests that dissolved oxygen originating from the oxidization of water cannot oxidize luminol producing the light. The decreased CL intensity became larger with increasing concentration of potassium chloride. Above the concentration of 0.080 M, it remained
Fig. 5. Effect of potassium chloride concentration on the decreased CL intensity for 1.010ÿ8 g mlÿ1 dopamine, 1.010ÿ5 M luminol, electrolysis current: 0.40 mA.
C. Zhang et al. / Analytica Chimica Acta 374 (1998) 105±110
109
obtained for dopamine, adrenaline and isoprenaline. The linear dynamic ranges, the correlation, detection limits and the relative standard deviations are summarized in Table 1. At a ¯ow rate of 2.0 ml minÿ1, the determination of analyte could be performed in 1 min including sampling and washing, giving a throughput of about 60 times per hour. 3.8. Application
Fig. 6. Effect of luminol concentration on the decreased CL intensity for 1.010ÿ8 g mlÿ1 dopamine, 0.10 M phosphate buffer containing 0.10 M potassium chloride (pH 10.0), electrolysis current: 0.40 mA.
almost constant. Thus the potassium chloride concentration of 0.10 M was chosen as optimum. 3.6. Effect of luminol concentration The in¯uence of the luminol concentration on the analytical signal was investigated. Fig. 6 shows the variation of the decreased CL intensity with the concentration of luminol solution. The greatest signal intensity was obtained when 1.010ÿ5 M luminol was used. In order to ensure maximum sensitivity, a luminol concentration of 1.010ÿ5 M was chosen in this work. 3.7. Calibration data Under the optimized condition and by use of the manifold depicted in Fig. 1, calibration curves were
In order to assess the possible application of the proposed method to the analysis of pharmaceutical dosage forms, the in¯uence of commonly used excipients and additives was investigated for the determination of 1.010ÿ8 g mlÿ1 catecholamines. No interference could be observed when the sample includes up to a 20-fold weight concentrations of fructose, glucose, lactose, sucrose, glycine, alanine, gum acacia, cellulose, or starch in the original catecholamines-containing powder. Additionally, the test also showed that more than 1000-fold excess of Ca2, Mg2, acetic acid, tartaric acid, citric acid, and less than 50-fold excess of uric acid, cholesterol, ascorbic acid did not interfere with the determination of 1.010ÿ8 g mlÿ1 catecholamines. Usually only one catecholamine is present in pharmaceutical preparations. The method may be suitable for adrenaline and isoprenaline determination in medicine. For biological ¯uids its applicability should be assessed carefully. The proposed method was applied to the analysis of some commercial injections. The results are shown in Table 2. As can be seen, there are no signi®cant differences between the labelled values and those obtained by the proposed method. In conclusion, the unstable reagents required for the CL reaction can be easily electrogenerated on-line and lead to methods for many potential applications. The proposed method for the determination of catechol-
Table 1 Parameters of the calibration curves used for the determination of dopamine, adrenaline and isoprenaline Catecholamine
Linear dynamic ranges (g mlÿ1)
Correlation coefficient (n5)
Detection limits (g mlÿ1)
RSD (%) (n11)
Dopamine Adrenaline Isoprenaline
110ÿ9±210ÿ8 210ÿ9±210ÿ8 210ÿ9±210ÿ8
0.9982 0.9975 0.9972
610ÿ10 110ÿ9 110ÿ9
2.4 2.8 2.6
110
C. Zhang et al. / Analytica Chimica Acta 374 (1998) 105±110
Table 2 Determination of three catecholamines in pharmaceutical preparations Preparations
Labelled value (g mlÿ1)
Present methoda found (g mlÿ1)
Recoverya (%)
RSDa (%)
Dopamine injection Adrenaline injection Isoprenaline injection
1.0 0.5 0.5
1.02 0.49 0.51
102 98 103
3.4 3.7 3.9
a
Average of three determinations.
amines by CL±FIA also demonstrates that CL has many potential applications in the area of pharmaceutical analysis. The method is superior to other conventional methods in that it is very fast, simple, and sensitive. Further work on this subject is in progress, aiming at liquid chromatographic applications. Acknowledgements This study was supported by the National Natural Science Foundation of China (no. 39730160). References [1] A.J. Pesce, L.A. Kaplan, S. Bircher (Ed.), Methods in Clinical Chemistry, C.V. Mosby, St. Louis, MO, 1987, p. 944. [2] J.J. Berzas Nevado, J.M. Lemus Gallego, P. Buitrago Laguna, Anal. Chim. Acta 300 (1995) 293.
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