Size-Dependent Active Effect of Cadmium Telluride Quantum Dots on Luminol–Potassium Periodate Chemiluminescence System for Levodopa Detection Jianbo Wang,a Lijuan Cui,b Suqin Han,b,* Fang Haob a b
Editorial Department of Journal, Shanxi Normal University, Linfen 041004, Shanxi, China School of Chemistry and Material Science, Shanxi Normal University, Linfen 041004, Shanxi, China
It was found that cadmium telluride (CdTe) quantum dots (QDs) with different sizes can have a great sensitizing effect on chemiluminescence (CL) emission from luminol–potassium periodate (KIO4) system. Levodopa, a widely prescribed drug in the treatment of Parkinson’s disease, could inhibit luminol–KIO4–CdTe QDs CL reaction in alkaline solution. The inhibited CL intensity was proportional to the concentration of levodopa in the range from 8.0 nM to 10.0 lM. The detection limit was 3.8 nM. This method has been successfully applied to determine levodopa in pharmaceutical preparation and human urine and plasma samples with recoveries of 94.1–105.4%. This was the first work for inhibition effect determination of levodopa using a QD-based CL method. Index Headings: Cadmium telluride; CdTe; Quantum dots; Chemiluminescence; Levodopa; Luminol; Pharmaceuticals.
INTRODUCTION Levodopa is a naturally occurring amino acid and a precursor of the neurotransmitter dopamine.1 Because dopamine does not cross the blood–brain barrier readily, it cannot be administered directly, while its precursor levodopa is given orally and is easily absorbed through the bowel and converted into dopamine by decarboxylase. Then, levodopa is used to increase dopamine in the brain, which reduces the symptoms of Parkinson’s disease. Levodopa can be slowly administered starting with one-fourth a tablet three times per day, which can be increased by one-quarter of a tablet per dose every week to optimum symptom relief up to a maximum of 1000–1200 mg per day.2 Nevertheless, elevated levels of dopamine could also cause adverse reactions such as nausea, vomiting, and cardiac arrhythmias.3 In order to achieve a better curative effect and a lower toxicity, it is very important to rapidly control the content of levodopa in biological fluids and pharmaceutical formulations. Various analytical methods have been proposed for the determination of levodopa, such as spectrophotometry,4 spectrofluorimetry,5,6 1H nuclear magnetic resonance spectroscopy,7 electrochemistry,8–10 chemiluminescence (CL),11 capillary electrophoresis,12–14 and high-performance liquid chromatography.15–17 The expensive instrumentation, time consumption, and low sensitivity were some of the reported shortcomings of these methods. The CL method has high potential for a great variety of Received 21 August 2014; accepted 5 January 2015. * Author to whom correspondence should be sent. E-mail: hsq@dns. sxnu.edu.cn. DOI: 10.1366/14-07632
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analytical applications due to its high sensitivity, wide linear range, and cost-effective apparatus because there is no need for an excitation light source and a spectral resolving system. However, CL analysis sometimes suffers from drawbacks, such as poor selectivity because of existing species in the solution that can alter analytical signals. The relatively low emission intensity of some CL reactions, due to possessing very low efficiency in transforming chemical energy into light, such as the potassium permanganate–sodium sulfite (KMnO4–Na2SO3) system for levodopa,11 is another possible shortcoming of CL-detection systems.18 Quantum dots (QDs), monodisperse crystalline semiconductor particles with a radius being less than or equal to the bulk-exciton Bohr radius, have attracted considerable attention in recent years because of their unique optical properties, such as photostability, bright fluorescence, size-dependent absorption and fluorescence, and narrow emission and broad absorption bands, as a result of the quantum confinement effect.19,20 The incorporation of QDs has opened up new ways in the field of novel applications with enhanced sensitivity, in which QDs participated in a CL reaction in emitting species21–23 or as catalysts.24 In the present work, we found that the levodopa could effectively quench the CL signal from the reaction of luminol–potassium periodate–cadmium telluride (KIO4– CdTe) QDs, and the decreased CL intensity was proportionate to the concentration of levodopa. On the basis of these findings, a simple and sensitive CL method was developed for the determination of levodopa in pharmaceutical formulation and human urine and plasma samples. Validity of the analytical results obtained from the proposed method was confirmed by comparing them with those obtained from a pharmacopeial method25 and by analyzing human urine and plasma spiked samples with satisfactory results.
EXPERIMENTAL Reagents. All the chemicals were of analyticalreagent grade and were used without further purification and prepared in double-distilled water. Luminol was purchased from Shaanxi Normal University (Shannxi, China). Levodopa was purchased from Chinese Pharmaceutical and Biological Test Institute (Beijing, China). Glutathione (GSH) and sodium borohydride (NaBH4) were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Cadmium nitrate tetrahydrate (Cd(NO3)24H2O), tellurium powder, KIO4, and absolute
0003-7028/15/6906-0699/0 Q 2015 Society for Applied Spectroscopy
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FIG. 1. Schematic diagram of the FI-CL system. (a) CdTe QD solution; (b) levodopa solution as the carrier; (c) luminol solution; (d) KIO4 solution; P, peristaltic pump; F, CL flow cell; PMT, photomultiplier tube; HV, negative high-voltage supply; PC, computer; W, waste solution.
alcohol were purchased from Tianjin Chemical Reagent Co. (Tianjin, China). Stock solution of KIO4 (0.01 mol/L) was prepared by dissolving KIO4 in water and stored in a refrigerator. Stock solution of levodopa (1.0 mM) was prepared by dissolving 19.72 mg levodopa with a 10 mL 0.1 mol/L HCl and diluting with water in a 100 mL volumetric flask. Stock solution of luminol (1.0 mM) was prepared by dissolving 177.16 mg luminol in 10 mL 0.1 mol/L NaOH and diluting with water in a 100 mL brown volumetric flask. Apparatus. An IFIS-C model flow injection system (Xi’an Remex Analysis Instrument Co. Ltd., China) was employed to deliver the solutions. The CL signal was collected with a BPCL model ultraweak luminescence analyzer (Institute of Biophysics Chinese Academy of Science, Beijing) and then recorded using a computer with BPCL software. Fluorescence (FL) measurements were performed on a Cary Eclipse fluorescence spectrophotometer. Ultraviolet (UV)-visible absorption spectra were obtained using a Cary 5000 spectrophotometer. Procedure. The flow injection–chemiluminescence (FI-CL) system procedure is shown in Fig. 1. Streams of 0.3 mM CdTe QDs, levodopa standard–sample solution (as carrier), a mixture of 6.0 lM luminol, 0.1M sodium hydroxide (NaOH), and 80.0 lM KIO4 were propelled into the flow lines using two peristaltic pumps, P1, a peristaltic pump at a flow rate of 2.0 mL/min, and P2, a peristaltic pump at 3.0 mL/min flow rate. The CdTe QD solution was injected into the carrier stream (levodopa standard–sample solution) using an eightway injection valve with a sample loop of 100 lL. The mixture stream was merged with the luminol solution at a Y-piece and then further mixed with KIO4 solution at another Y-piece. The mixed solution stream was delivered into the flow cell. The CL signal produced in the flow cell was detected and amplified by the photomultiplier tube and luminometer, and then imported to the computer for data acquisition. As mentioned above, levodopa was found to obviously inhibit the CL signal of the luminol–KIO4–CdTe QD system. Determination of levodopa was based on the CL intensity changes from the without and with levodopa sample solutions. The net CL intensity of DI = I0 – I was proportional to the
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concentration of levodopa, where I0 and I denoted CL intensity in the absence and presence of levodopa, respectively. Synthesis of Cadmium Telluride Quantum Dots Capped with Glutathione. As cited in the literature,26 the synthesis of CdTe QDs with GSH as a stabilizer was less time consuming, and the obtained QDs exhibit strong FL with high quantum yield compared to the conventional organometallic approaches. So we adopted this previously reported method with little modification. Briefly, sodium hydrogen telluride (NaHTe) was prepared by adding 200 mg NaBH4 to a flask containing 25.4 mg tellurium powder and 10 mL water under a N2 atmosphere. The reaction was continued until all of the tellurium powder was dissolved. Cd(NO3)2 (154.2 mg) and GSH (184.2 mg) were dissolved in 93 mL water, followed by adjustment of the pH to 11.5 with 1.0 mol/L NaOH solution. The mixture was deaerated by N2 bubbling for 30 min. Then, freshly prepared NaHTe solution (0.1 mmol) was quickly injected into the mixture under vigorous stirring, followed by refluxing the reaction mixture for 60, 90, and 120 min at 95 8C. The final concentration of QDs was 1.0 mM according to the Te2– concentration and then kept at 4 8C in the dark for later use. Cadmium telluride QD samples used in the present work were purified by selective precipitation with absolute alcohol and centrifuged for 10 min at 5000 rpm. The residue was redispersed in ultrapure water for subsequent research.
RESULT AND DISCUSSION Ultraviolet–Visible and Fluorescence Spectra and the Sensitized Effect on the Chemiluminescence of Cadmium Telluride Quantum Dots. Figure 2 shows the UV-Vis absorption spectra (a), FL spectra (b), and the sensitized effect on CL of CdTe QDs (c). These CdTe QDs had an absorption maximum of the first electronic transition, and the FL peak and absorption maximum shifted to longer wavelengths with increasing QD diameter. The diameter of CdTe QDs, which were calculated by Peng’s method,27 were around 2.6, 3.2, and 3.4 nm, respectively, corresponding with the FL peaks of 539, 562, and 583 nm. The effects of CdTe QDs on the luminol–KIO4 CL system were investigated as shown in the procedure in Fig. 1, in which the carrier of levodopa changed to water. As shown in Fig. 2c, the CL emission from the luminol– KIO4 CL system was relatively weak. However, with the incorporation of CdTe QDs, CL intensity was enhanced by the 2.6, 3.2, and 3.4 nm diameter CdTe QDs, and the most intensive CL signal was obtained with the CdTe QDs that had a diameter of 3.2 nm. Thus, 3.2 nm CdTe QDs were chosen in the following experiments. Inhibition of Levodopa on the Cadmium Telluride Quantum Dot-Enhanced Chemiluminescence Assay. The effects of levodopa on CdTe QD-enhanced CL were studied. Figure 3 showed the kinetic curve of the CL system in static injection mode. The addition of CdTe QDs into the luminol–KIO4 system could greatly enhance on CL signals. It was found that the present reaction went fast; about 5 s was needed for the maximum peak to
FIG. 2. (a) UV-Vis spectra, (b) FL spectra, and (c) CL profiles with or without CdTe QDs. Conditions: luminol, 6.0 lM; KIO4, 80.0 lM; NaOH, 0.1 M; CdTe QDs, 0.1 mM; levodopa, 8.0 lM.
appear, and it took 12 s for the signal to decline to the basement. In the presence of levodopa, the intensity of CdTe QD-enhanced CL was declined. Therefore, the CdTe QD-enhanced CL system was chosen to establish the method for the determination of levodopa in pharmaceutical preparations. The strongest inhibition was obtained by optimizing the CL parameters. Optimization of the Chemiluminescence Parameters. Different parameters were investigated systematically to establish optimum conditions for the
FIG. 3. Kinetic curves of CL in static injection mode. (a) luminol–KIO4; (b) luminol–KIO4–CdTe QD-levodopa; (c) luminol–KIO4–CdTe QDs (purifying); (d) luminol–KIO4–CdTe QDs (no purifying).
determination of levodopa (Fig. 4). In this CL system, luminol concentration had a significant influence on the CL assay. Fixing the KIO4 concentration at 80.0 lM, NaOH concentration at 0.1 M, and CdTe QD concentration at 0.3 mM, the effect of the luminol concentration on the increased CL intensity was examined in the range of 1.0 to 8.0 lM. It was found that the maximum DI was reached when luminol concentration was 6.0 lM. So the luminol concentration of 6.0 lM was chosen for subsequent research work. The effect of KIO4 concentration on the signal intensity was examined ranging from 40.0 to 100.0 lM keeping the luminol concentration at 6.0 lM, NaOH concentration at 0.1 M, and CdTe QD concentration at 0.3 mM. The results showed that the DI increased from 40.0 to 80.0 lM and then decreased from 80.0 lM. As a result, the concentration of 80.0 lM KIO4 was chosen for levodopa determination. The CL reaction of luminol with KIO4 was performed in an alkaline medium. Therefore, NaOH was added in luminol solution to improve the sensitivity of reaction. Maximum DI was obtained with the 0.1 M NaOH in luminol solution when the 6.0 lM luminol, 80.0 lM KIO4, and 0.3 mM CdTe QDs were used. The optimized concentration of NaOH was therefore chosen as 0.1 M. Fixing the luminol concentration at 6.0 lM, KIO4 concentration at 80.0 lM, and NaOH concentration at 0.1 M, the effect of concentration of CdTe QDs was also studied. The CL intensity was gradually increased as the concentration of CdTe QDs increased over the range 0.1–0.3 mM. However, if the concentration of CdTe QDs was more than 0.3 mM, the CL intensity decreased instead. Therefore, the optimum
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FIG. 4.
Influence of the concentration of (a) luminol, (b) KIO4, (c) NaOH, and (d) CdTe NPs on the CL intensity.
CdTe QD concentration was chosen to be 0.3 mM. Furthermore, flow rate was an important factor in the FICL system. An optimum flow rate was necessary for the maximum collection of emitted light in the flow cell. In the range of 1.5–3.5 mL/min, a flow rate of 3.0 mL/min was chosen for subsequent work considering the stability of instruments and low reagent consumption. After a careful study on the effects of the above several parameters, the CL conditions for the determination of levodopa were selected as the following: 6.0 lM luminol in 0.1 M NaOH solution; 80.0 lM KIO4, 0.3 Mm CdTe QDs, and 3.0 mL/min flow rate of the peristaltic pump. Working Curve and Detection Limit. A calibration curve was obtained for the determination of levodopa under the optimal experimental conditions. It gave a linear range of 8.0 nM–10.0 lM. The linear calibration response curves can be described by the equation DI = 49 650.7c þ 736.9 (c = 10.0 lM, R = 0.997). The calibration curve for the determination of levodopa is shown in Fig. 5. The limit of detection (LOD) as defined by the International Union of Pure and Applied Chemistry was found to be 3.8 nM. The relative standard deviation (RSD) for 11 repeated determinations of 5.0 lM levodopa was 2.4%. Interference. The influence of some possibly coexisting inorganic and organic ions on the CL intensity was investigated for determining levodopa by comparing with the CL emissions obtained using analyte solution alone or with foreign species added. The tolerable limit of foreign species was taken as a relative error not greater than 65% in the recovery at a concentration of 1.0 lM levodopa solution. The tolerance limit was taken as the amount that caused a relative error of 65% in the peak height. The tolerated ratios of foreign substances to analyte were 1000-fold for Naþ, Cl–, CO32–, and NO3–; 800-
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fold for Br–, Zn2þ, HCO3–, starch, and dextrin; 500-fold for NH4þ, EDTA, magnesium stearate, and sucrose; 250-fold for Ac–, Mn2þ, Cu2þ, leucine, ascorbic acid, and citric acid; 100-fold for tryptophan, glucose, and lysine; 80-fold for Mg2þ, Al3þ, phenylalanine, and sarcosine; 50-fold for Ni2þ, Co2þ, acetonitrile, and albumin; 20-fold for Fe3þ, dextrin, and lactic acid had no interference on the determination of levodopa. Application of the Proposed Method. Ten levodopa tablets were weighed and powdered in a mortar. The powder of 0.25 g (equivalent to the weight of one tablet) was dissolved in 10 mL 0.1 M HCl solution. The solution was centrifuged at 2000 rpm for 10 min, and the supernatant was transferred into a 25 mL volumetric flask and diluted to the mark with water to obtain the levodopa sample solution. The sample solution was further diluted with water to bring levodopa concentrations within the linear range and then analyzed according to the procedure described above. Results are summarized in Table I. The t-test assumed that there is no significant difference between the results obtained by the proposed method and those obtained by the Chinese pharmacopeia method25 at the confidence level of 95%. Recoveries were in the range of 94.1–103.3%. The proposed method was applied to determine levodopa in spiked human urine and plasma samples. These samples were supplied by two healthy volunteers. An aliquot of standard levodopa solution was added to 1 mL of urine or plasma in a centrifuge tube and mixed for 20 min, respectively. Here, 2 mL of acetonitrile was added for deproteination. It was blended on a vortex mixer and centrifuged at 5000 rpm for 10 min. The protein-free supernatant was transferred into a volumetric flask and evaporated to dryness under a stream of nitrogen at room temperature. The dry residue was
FIG. 5. Calibration curves for the determination of levodopa in the range 8.0 nM–0.2 lM (inset) and 8.0 nM–10.0 lM. TABLE I. Results of the determination of levodopa in tablets (n = 5).
Samples 1 2 3
Nominal content (g/tablet)
Proposed method
Recovery (%)
RSD (%)
Pharmacopoeia method (g/tablet)
RSD (%)
0.25 0.25 0.25
0.2618 0.2620 0.2623
103.3 98.4 94.1
2.23 2.09 1.99
0.2616 0.2619 0.2620
1.85 1.98 2.27
TABLE II. Results of the determination of levodopa in urine and plasma (n = 5).
Samples Urine 1
Concentration (0.1 lM) 1.0
Urine 2
1.0
Plasma 1
1.0
Plasma 2
1.0
Added (0.1 lM)
Founded (0.1 lM)
0 1.0 0 1.0 0 1.0 0 1.0
0.923 1.949 1.031 2.085 1.065 2.047 0.977 1.918
dissolved and diluted to 10 mL with water, and we then proceeded as described above. A blank value was determined by treating drug-free urine or plasma in the same way. Recovery was determined by comparing the representative peak height of the urine or plasma sample with the peak height of the standard drug at the same concentration. The obtained results were given in Table II. For urine and plasma samples, the recoveries of the drug were in the range of 94.1–105.4%. The suitability question of using the calibration curve for quantitative analysis of the biological samples was evaluated by
Recovery (%) 102.6 105.4 98.2 94.1
RSD (%) 2.22 2.07 1.91 1.98 2.42 2.35 2.11 1.95
RSD. The result showed that the RSD was 2.1% in intraday, and in interday was 4.2%.
CONCLUSION In this study, the CL signal from the luminol–KIO4– CdTe QD system was strongly inhibited by the levodopa. Based on this inhibition, a novel CL method was developed for the determination of levodopa in pharmaceutical preparations and human urine and plasma samples. The proposed method did not require tedious
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pretreatment or sophisticated equipment, but rather simplicity, sensitivity, and accuracy. The results compared well with the pharmacopeia method.
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ACKNOWLEDGMENTS This work was financially supported by the Natural Science Foundation of Shanxi (Grant No. 2013011013-3), Undergraduate Training Programs for Innovation and Entrepreneurship of Shanxi Normal University (SD2014CXXM-50), and Shanxi Normal University of Modern Arts and Sciences (WL2014CXCY-10), Teaching Reform Project of Shanxi Normal University (SD2013JGXM-55). All of the authors express their deep thanks.
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Editorial Department of Journal, Shanxi Normal University, Linfen 041004, Shanxi, China School of Chemistry and Material Science, Shanxi Normal University, Linfen 041004, Shanxi, China
It was found that cadmium telluride (CdTe) quantum dots (QDs) with different sizes can have a great sensitizing effect on chemiluminescence (CL) emission from luminol–potassium periodate (KIO4) system. Levodopa, a widely prescribed drug in the treatment of Parkinson’s disease, could inhibit luminol–KIO4–CdTe QDs CL reaction in alkaline solution. The inhibited CL intensity was proportional to the concentration of levodopa in the range from 8.0 nM to 10.0 lM. The detection limit was 3.8 nM. This method has been successfully applied to determine levodopa in pharmaceutical preparation and human urine and plasma samples with recoveries of 94.1–105.4%. This was the first work for inhibition effect determination of levodopa using a QD-based CL method. Index Headings: Cadmium telluride; CdTe; Quantum dots; Chemiluminescence; Levodopa; Luminol; Pharmaceuticals.
INTRODUCTION Levodopa is a naturally occurring amino acid and a precursor of the neurotransmitter dopamine.1 Because dopamine does not cross the blood–brain barrier readily, it cannot be administered directly, while its precursor levodopa is given orally and is easily absorbed through the bowel and converted into dopamine by decarboxylase. Then, levodopa is used to increase dopamine in the brain, which reduces the symptoms of Parkinson’s disease. Levodopa can be slowly administered starting with one-fourth a tablet three times per day, which can be increased by one-quarter of a tablet per dose every week to optimum symptom relief up to a maximum of 1000–1200 mg per day.2 Nevertheless, elevated levels of dopamine could also cause adverse reactions such as nausea, vomiting, and cardiac arrhythmias.3 In order to achieve a better curative effect and a lower toxicity, it is very important to rapidly control the content of levodopa in biological fluids and pharmaceutical formulations. Various analytical methods have been proposed for the determination of levodopa, such as spectrophotometry,4 spectrofluorimetry,5,6 1H nuclear magnetic resonance spectroscopy,7 electrochemistry,8–10 chemiluminescence (CL),11 capillary electrophoresis,12–14 and high-performance liquid chromatography.15–17 The expensive instrumentation, time consumption, and low sensitivity were some of the reported shortcomings of these methods. The CL method has high potential for a great variety of Received 21 August 2014; accepted 5 January 2015. * Author to whom correspondence should be sent. E-mail: hsq@dns. sxnu.edu.cn. DOI: 10.1366/14-07632
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analytical applications due to its high sensitivity, wide linear range, and cost-effective apparatus because there is no need for an excitation light source and a spectral resolving system. However, CL analysis sometimes suffers from drawbacks, such as poor selectivity because of existing species in the solution that can alter analytical signals. The relatively low emission intensity of some CL reactions, due to possessing very low efficiency in transforming chemical energy into light, such as the potassium permanganate–sodium sulfite (KMnO4–Na2SO3) system for levodopa,11 is another possible shortcoming of CL-detection systems.18 Quantum dots (QDs), monodisperse crystalline semiconductor particles with a radius being less than or equal to the bulk-exciton Bohr radius, have attracted considerable attention in recent years because of their unique optical properties, such as photostability, bright fluorescence, size-dependent absorption and fluorescence, and narrow emission and broad absorption bands, as a result of the quantum confinement effect.19,20 The incorporation of QDs has opened up new ways in the field of novel applications with enhanced sensitivity, in which QDs participated in a CL reaction in emitting species21–23 or as catalysts.24 In the present work, we found that the levodopa could effectively quench the CL signal from the reaction of luminol–potassium periodate–cadmium telluride (KIO4– CdTe) QDs, and the decreased CL intensity was proportionate to the concentration of levodopa. On the basis of these findings, a simple and sensitive CL method was developed for the determination of levodopa in pharmaceutical formulation and human urine and plasma samples. Validity of the analytical results obtained from the proposed method was confirmed by comparing them with those obtained from a pharmacopeial method25 and by analyzing human urine and plasma spiked samples with satisfactory results.
EXPERIMENTAL Reagents. All the chemicals were of analyticalreagent grade and were used without further purification and prepared in double-distilled water. Luminol was purchased from Shaanxi Normal University (Shannxi, China). Levodopa was purchased from Chinese Pharmaceutical and Biological Test Institute (Beijing, China). Glutathione (GSH) and sodium borohydride (NaBH4) were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Cadmium nitrate tetrahydrate (Cd(NO3)24H2O), tellurium powder, KIO4, and absolute
0003-7028/15/6906-0699/0 Q 2015 Society for Applied Spectroscopy
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FIG. 1. Schematic diagram of the FI-CL system. (a) CdTe QD solution; (b) levodopa solution as the carrier; (c) luminol solution; (d) KIO4 solution; P, peristaltic pump; F, CL flow cell; PMT, photomultiplier tube; HV, negative high-voltage supply; PC, computer; W, waste solution.
alcohol were purchased from Tianjin Chemical Reagent Co. (Tianjin, China). Stock solution of KIO4 (0.01 mol/L) was prepared by dissolving KIO4 in water and stored in a refrigerator. Stock solution of levodopa (1.0 mM) was prepared by dissolving 19.72 mg levodopa with a 10 mL 0.1 mol/L HCl and diluting with water in a 100 mL volumetric flask. Stock solution of luminol (1.0 mM) was prepared by dissolving 177.16 mg luminol in 10 mL 0.1 mol/L NaOH and diluting with water in a 100 mL brown volumetric flask. Apparatus. An IFIS-C model flow injection system (Xi’an Remex Analysis Instrument Co. Ltd., China) was employed to deliver the solutions. The CL signal was collected with a BPCL model ultraweak luminescence analyzer (Institute of Biophysics Chinese Academy of Science, Beijing) and then recorded using a computer with BPCL software. Fluorescence (FL) measurements were performed on a Cary Eclipse fluorescence spectrophotometer. Ultraviolet (UV)-visible absorption spectra were obtained using a Cary 5000 spectrophotometer. Procedure. The flow injection–chemiluminescence (FI-CL) system procedure is shown in Fig. 1. Streams of 0.3 mM CdTe QDs, levodopa standard–sample solution (as carrier), a mixture of 6.0 lM luminol, 0.1M sodium hydroxide (NaOH), and 80.0 lM KIO4 were propelled into the flow lines using two peristaltic pumps, P1, a peristaltic pump at a flow rate of 2.0 mL/min, and P2, a peristaltic pump at 3.0 mL/min flow rate. The CdTe QD solution was injected into the carrier stream (levodopa standard–sample solution) using an eightway injection valve with a sample loop of 100 lL. The mixture stream was merged with the luminol solution at a Y-piece and then further mixed with KIO4 solution at another Y-piece. The mixed solution stream was delivered into the flow cell. The CL signal produced in the flow cell was detected and amplified by the photomultiplier tube and luminometer, and then imported to the computer for data acquisition. As mentioned above, levodopa was found to obviously inhibit the CL signal of the luminol–KIO4–CdTe QD system. Determination of levodopa was based on the CL intensity changes from the without and with levodopa sample solutions. The net CL intensity of DI = I0 – I was proportional to the
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concentration of levodopa, where I0 and I denoted CL intensity in the absence and presence of levodopa, respectively. Synthesis of Cadmium Telluride Quantum Dots Capped with Glutathione. As cited in the literature,26 the synthesis of CdTe QDs with GSH as a stabilizer was less time consuming, and the obtained QDs exhibit strong FL with high quantum yield compared to the conventional organometallic approaches. So we adopted this previously reported method with little modification. Briefly, sodium hydrogen telluride (NaHTe) was prepared by adding 200 mg NaBH4 to a flask containing 25.4 mg tellurium powder and 10 mL water under a N2 atmosphere. The reaction was continued until all of the tellurium powder was dissolved. Cd(NO3)2 (154.2 mg) and GSH (184.2 mg) were dissolved in 93 mL water, followed by adjustment of the pH to 11.5 with 1.0 mol/L NaOH solution. The mixture was deaerated by N2 bubbling for 30 min. Then, freshly prepared NaHTe solution (0.1 mmol) was quickly injected into the mixture under vigorous stirring, followed by refluxing the reaction mixture for 60, 90, and 120 min at 95 8C. The final concentration of QDs was 1.0 mM according to the Te2– concentration and then kept at 4 8C in the dark for later use. Cadmium telluride QD samples used in the present work were purified by selective precipitation with absolute alcohol and centrifuged for 10 min at 5000 rpm. The residue was redispersed in ultrapure water for subsequent research.
RESULT AND DISCUSSION Ultraviolet–Visible and Fluorescence Spectra and the Sensitized Effect on the Chemiluminescence of Cadmium Telluride Quantum Dots. Figure 2 shows the UV-Vis absorption spectra (a), FL spectra (b), and the sensitized effect on CL of CdTe QDs (c). These CdTe QDs had an absorption maximum of the first electronic transition, and the FL peak and absorption maximum shifted to longer wavelengths with increasing QD diameter. The diameter of CdTe QDs, which were calculated by Peng’s method,27 were around 2.6, 3.2, and 3.4 nm, respectively, corresponding with the FL peaks of 539, 562, and 583 nm. The effects of CdTe QDs on the luminol–KIO4 CL system were investigated as shown in the procedure in Fig. 1, in which the carrier of levodopa changed to water. As shown in Fig. 2c, the CL emission from the luminol– KIO4 CL system was relatively weak. However, with the incorporation of CdTe QDs, CL intensity was enhanced by the 2.6, 3.2, and 3.4 nm diameter CdTe QDs, and the most intensive CL signal was obtained with the CdTe QDs that had a diameter of 3.2 nm. Thus, 3.2 nm CdTe QDs were chosen in the following experiments. Inhibition of Levodopa on the Cadmium Telluride Quantum Dot-Enhanced Chemiluminescence Assay. The effects of levodopa on CdTe QD-enhanced CL were studied. Figure 3 showed the kinetic curve of the CL system in static injection mode. The addition of CdTe QDs into the luminol–KIO4 system could greatly enhance on CL signals. It was found that the present reaction went fast; about 5 s was needed for the maximum peak to
FIG. 2. (a) UV-Vis spectra, (b) FL spectra, and (c) CL profiles with or without CdTe QDs. Conditions: luminol, 6.0 lM; KIO4, 80.0 lM; NaOH, 0.1 M; CdTe QDs, 0.1 mM; levodopa, 8.0 lM.
appear, and it took 12 s for the signal to decline to the basement. In the presence of levodopa, the intensity of CdTe QD-enhanced CL was declined. Therefore, the CdTe QD-enhanced CL system was chosen to establish the method for the determination of levodopa in pharmaceutical preparations. The strongest inhibition was obtained by optimizing the CL parameters. Optimization of the Chemiluminescence Parameters. Different parameters were investigated systematically to establish optimum conditions for the
FIG. 3. Kinetic curves of CL in static injection mode. (a) luminol–KIO4; (b) luminol–KIO4–CdTe QD-levodopa; (c) luminol–KIO4–CdTe QDs (purifying); (d) luminol–KIO4–CdTe QDs (no purifying).
determination of levodopa (Fig. 4). In this CL system, luminol concentration had a significant influence on the CL assay. Fixing the KIO4 concentration at 80.0 lM, NaOH concentration at 0.1 M, and CdTe QD concentration at 0.3 mM, the effect of the luminol concentration on the increased CL intensity was examined in the range of 1.0 to 8.0 lM. It was found that the maximum DI was reached when luminol concentration was 6.0 lM. So the luminol concentration of 6.0 lM was chosen for subsequent research work. The effect of KIO4 concentration on the signal intensity was examined ranging from 40.0 to 100.0 lM keeping the luminol concentration at 6.0 lM, NaOH concentration at 0.1 M, and CdTe QD concentration at 0.3 mM. The results showed that the DI increased from 40.0 to 80.0 lM and then decreased from 80.0 lM. As a result, the concentration of 80.0 lM KIO4 was chosen for levodopa determination. The CL reaction of luminol with KIO4 was performed in an alkaline medium. Therefore, NaOH was added in luminol solution to improve the sensitivity of reaction. Maximum DI was obtained with the 0.1 M NaOH in luminol solution when the 6.0 lM luminol, 80.0 lM KIO4, and 0.3 mM CdTe QDs were used. The optimized concentration of NaOH was therefore chosen as 0.1 M. Fixing the luminol concentration at 6.0 lM, KIO4 concentration at 80.0 lM, and NaOH concentration at 0.1 M, the effect of concentration of CdTe QDs was also studied. The CL intensity was gradually increased as the concentration of CdTe QDs increased over the range 0.1–0.3 mM. However, if the concentration of CdTe QDs was more than 0.3 mM, the CL intensity decreased instead. Therefore, the optimum
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FIG. 4.
Influence of the concentration of (a) luminol, (b) KIO4, (c) NaOH, and (d) CdTe NPs on the CL intensity.
CdTe QD concentration was chosen to be 0.3 mM. Furthermore, flow rate was an important factor in the FICL system. An optimum flow rate was necessary for the maximum collection of emitted light in the flow cell. In the range of 1.5–3.5 mL/min, a flow rate of 3.0 mL/min was chosen for subsequent work considering the stability of instruments and low reagent consumption. After a careful study on the effects of the above several parameters, the CL conditions for the determination of levodopa were selected as the following: 6.0 lM luminol in 0.1 M NaOH solution; 80.0 lM KIO4, 0.3 Mm CdTe QDs, and 3.0 mL/min flow rate of the peristaltic pump. Working Curve and Detection Limit. A calibration curve was obtained for the determination of levodopa under the optimal experimental conditions. It gave a linear range of 8.0 nM–10.0 lM. The linear calibration response curves can be described by the equation DI = 49 650.7c þ 736.9 (c = 10.0 lM, R = 0.997). The calibration curve for the determination of levodopa is shown in Fig. 5. The limit of detection (LOD) as defined by the International Union of Pure and Applied Chemistry was found to be 3.8 nM. The relative standard deviation (RSD) for 11 repeated determinations of 5.0 lM levodopa was 2.4%. Interference. The influence of some possibly coexisting inorganic and organic ions on the CL intensity was investigated for determining levodopa by comparing with the CL emissions obtained using analyte solution alone or with foreign species added. The tolerable limit of foreign species was taken as a relative error not greater than 65% in the recovery at a concentration of 1.0 lM levodopa solution. The tolerance limit was taken as the amount that caused a relative error of 65% in the peak height. The tolerated ratios of foreign substances to analyte were 1000-fold for Naþ, Cl–, CO32–, and NO3–; 800-
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fold for Br–, Zn2þ, HCO3–, starch, and dextrin; 500-fold for NH4þ, EDTA, magnesium stearate, and sucrose; 250-fold for Ac–, Mn2þ, Cu2þ, leucine, ascorbic acid, and citric acid; 100-fold for tryptophan, glucose, and lysine; 80-fold for Mg2þ, Al3þ, phenylalanine, and sarcosine; 50-fold for Ni2þ, Co2þ, acetonitrile, and albumin; 20-fold for Fe3þ, dextrin, and lactic acid had no interference on the determination of levodopa. Application of the Proposed Method. Ten levodopa tablets were weighed and powdered in a mortar. The powder of 0.25 g (equivalent to the weight of one tablet) was dissolved in 10 mL 0.1 M HCl solution. The solution was centrifuged at 2000 rpm for 10 min, and the supernatant was transferred into a 25 mL volumetric flask and diluted to the mark with water to obtain the levodopa sample solution. The sample solution was further diluted with water to bring levodopa concentrations within the linear range and then analyzed according to the procedure described above. Results are summarized in Table I. The t-test assumed that there is no significant difference between the results obtained by the proposed method and those obtained by the Chinese pharmacopeia method25 at the confidence level of 95%. Recoveries were in the range of 94.1–103.3%. The proposed method was applied to determine levodopa in spiked human urine and plasma samples. These samples were supplied by two healthy volunteers. An aliquot of standard levodopa solution was added to 1 mL of urine or plasma in a centrifuge tube and mixed for 20 min, respectively. Here, 2 mL of acetonitrile was added for deproteination. It was blended on a vortex mixer and centrifuged at 5000 rpm for 10 min. The protein-free supernatant was transferred into a volumetric flask and evaporated to dryness under a stream of nitrogen at room temperature. The dry residue was
FIG. 5. Calibration curves for the determination of levodopa in the range 8.0 nM–0.2 lM (inset) and 8.0 nM–10.0 lM. TABLE I. Results of the determination of levodopa in tablets (n = 5).
Samples 1 2 3
Nominal content (g/tablet)
Proposed method
Recovery (%)
RSD (%)
Pharmacopoeia method (g/tablet)
RSD (%)
0.25 0.25 0.25
0.2618 0.2620 0.2623
103.3 98.4 94.1
2.23 2.09 1.99
0.2616 0.2619 0.2620
1.85 1.98 2.27
TABLE II. Results of the determination of levodopa in urine and plasma (n = 5).
Samples Urine 1
Concentration (0.1 lM) 1.0
Urine 2
1.0
Plasma 1
1.0
Plasma 2
1.0
Added (0.1 lM)
Founded (0.1 lM)
0 1.0 0 1.0 0 1.0 0 1.0
0.923 1.949 1.031 2.085 1.065 2.047 0.977 1.918
dissolved and diluted to 10 mL with water, and we then proceeded as described above. A blank value was determined by treating drug-free urine or plasma in the same way. Recovery was determined by comparing the representative peak height of the urine or plasma sample with the peak height of the standard drug at the same concentration. The obtained results were given in Table II. For urine and plasma samples, the recoveries of the drug were in the range of 94.1–105.4%. The suitability question of using the calibration curve for quantitative analysis of the biological samples was evaluated by
Recovery (%) 102.6 105.4 98.2 94.1
RSD (%) 2.22 2.07 1.91 1.98 2.42 2.35 2.11 1.95
RSD. The result showed that the RSD was 2.1% in intraday, and in interday was 4.2%.
CONCLUSION In this study, the CL signal from the luminol–KIO4– CdTe QD system was strongly inhibited by the levodopa. Based on this inhibition, a novel CL method was developed for the determination of levodopa in pharmaceutical preparations and human urine and plasma samples. The proposed method did not require tedious
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pretreatment or sophisticated equipment, but rather simplicity, sensitivity, and accuracy. The results compared well with the pharmacopeia method.
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ACKNOWLEDGMENTS This work was financially supported by the Natural Science Foundation of Shanxi (Grant No. 2013011013-3), Undergraduate Training Programs for Innovation and Entrepreneurship of Shanxi Normal University (SD2014CXXM-50), and Shanxi Normal University of Modern Arts and Sciences (WL2014CXCY-10), Teaching Reform Project of Shanxi Normal University (SD2013JGXM-55). All of the authors express their deep thanks.
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