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Determination of atropine using Mn-doped ZnS quantum dots as novel luminescent sensitizers
Journal of Luminescence 144 (2013) 34–40
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Determination of atropine using Mn-doped ZnS quantum dots as novel luminescent sensitizers Seyed Naser Azizi a, Mohammad Javad Chaichi a,n, Parmis Shakeri a, Ahmadreza Bekhradnia b a b
Analytical Division, Faculty of Chemistry, University of Mazandaran, Babolsar 4741695447, Iran Pharmaceutical Sciences Research Center, Department of Medicinal Chemistry, Mazandaran University of Medical Sciences, Sari, Iran
art ic l e i nf o
a b s t r a c t
Article history: Received 16 March 2013 Received in revised form 8 May 2013 Accepted 30 May 2013 Available online 26 June 2013
A novel chemiluminescence (CL) method using water-soluble Mn-doped ZnS quantum dots (QDs) as sensitizers is proposed for the chemiluminometric determination of atropine in pharmaceutical formulation. Water-soluble Mn-doped ZnS QDs were synthesized by using L-cysteine as stabilizer in aqueous solutions. The nanoparticles were structurally and optically characterized by X-ray powder diffraction (XRD), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), UV–vis absorption spectroscopy and photoluminescence (PL) emission spectroscopy. It was found that ZnS quantum dots acted as enhancers of the weak CL emission produced upon oxidation of sulfite by Ce(IV) in acidic medium. Trace amounts of atropine improved the sensitize effect of ZnS quantum dots yielding a significant chemiluminescence enhancement of the Ce(IV)–SO32−–ZnS QD system. Therefore, a new CL analysis system was developed for the determination of atropine. Under the optimum conditions, there is a good linear relationship between the relative chemiluminescence intensity and the concentration of atropine in the range of 1 10−9–1 10−6 M of atropine with a correlation coefficient (R2) of 0.9992. The limit of detection of this system was found to be 2.54 10−10 M. This method is not only simple, sensitive and low cost, but also reliable for practical applications. & 2013 Elsevier B.V. All rights reserved.
Keywords: ZnS quantum dot Sensitized chemiluminescence Ce(IV)–sulfite Atropine
1. Introduction Atropine (Fig. 1) is an alkaloid, which is mainly obtained from Atropa belladonna and in much smaller quantities from Datura stamonium [1]. It has been commonly used in pharmaceutical preparations for the treatment of gastrointestinal diseases, cardiopathy and parkinsonism [2]. Atropine is also used to dilate the pupils in eye operations. The most specific use of atropine is an antidote to organophosphate cholinesterase inhibitors, found in certain insecticides and chemical warfare nerve gases [3]. A high dosage of atropine also stimulates the central nerve system. Despite these positive effects of atropine against diseases, it is also a highly toxic compound. For instance, abnormal kidney function may lead to a toxic reaction in patients receiving atropine treatment, therefore the dosage level of atropine in pharmaceutical preparation is generally very low and for this reason it demands sensitive and selective detection techniques. Several analytical methods have been developed for the analysis of atropine in pharmaceutical dosage forms and in biological
n
Corresponding author. Tel./fax: +98 112 534 2350. E-mail address: [email protected] (M.J. Chaichi).
0022-2313/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jlumin.2013.05.054
fluids including liquid chromatography (LC) with UV detector [4] or tandem mass spectrometry [5], electrochemical method [6], post-chemiluminescence (PCL) [7], electrochemiluminescence (ECL) [8] and capillary electrophoresis [9]. As the dosage level of atropine in pharmaceutical preparation is generally very low and its quantitative determination is significant not only in clinical application but also pharmaceutical analysis; it is important to develop simple, sensitive and accurate methods for being able to detect atropine in formulations and biological systems. During the past decade, semiconductor nanocrystals (quantum dots, QDs) have attracted great interest due to their unique optical and electronic properties, originating from their large surface-tovolume ratio and confinement effect, and have proved to be promising in a number of application areas including biological markers [10], light-emitting diodes [11] or solar cells [12]. One important subset of QDs is those doped with a small percentage of dopants to alter their electronic, magnetic and optical properties for various desired applications [13,14]. Recent investigations have evaluated the use of metallic dopants with quantum states remote from the valence and conduction band edges of the semiconductor nanoparticles to generate different radiative processes [15,16]. In this related, Mn-doped ZnS QDs have attracted considerable attention lately
S.N. Azizi et al. / Journal of Luminescence 144 (2013) 34–40
Fig. 1. Chemical structure of atropine.
because doping Mn2+ ions may act as recombination centers for the excited electron–hole pairs and result in strong and characteristic luminescence at longer wavelengths. Mn2+ emission depends on many parameters like radial location of the dopant, surface ligand and surface charge. So upon Mn2+ doping, a characteristic emission band centered at around 570 nm with a neutral ligand to 600 nm for a charged one, is developed for the well-known 4 T1–6A1 d–d transition of Mn2+ ions on Zn2+ sites of the QD (where Mn2+ is coordinated by S2−) [17,18]. These doped QDs (d-dots) having longer luminescence excited state lifetime, minimized selfabsorption and high quantum efficiency [19] that can be used as cadmium-free emitters for biolabeling [10], as efficient donors for the Förster resonance energy transfer [20], or as sensors for organic and inorganic compounds [21]. In addition the optical properties of QDs strongly depend on the nature of their surface thus modifications of the surface with functional groups or biomolecules and the interactions that it could establish with specific analytes can result in dramatic changes in these properties [22]. Therefore, fluorescence or chemiluminescence (CL) based chemical sensing involving QDs have been developed for different chemical species such as ascorbic acid [23], urea [24], folic acid [25], acetone [26] and cysteine [27]. In most QDs applications, the detection is based on signal quenching, while a more newly attention has been focused on signal enhancing, mainly related to QD ability to sensitize different chemiluminescent systems. Sensitized chemiluminescence is an expeditious policy to exploit CL reactions with low quantum efficiencies for analytical purposes. The weak created energy is transferred to a sensitizer, usually an organic fluorophore with high quantum yield, which is able to magnify it. Any species that selectively interacts with the fluorophore could quench or enhance the CL emission. There are only a few reports involving the sensitized chemiluminescence of semiconductor nanocrystals [23,27–31]. To our knowledge, up to know, there are only few reports on the sensitize effect of ZnS QDs on distinct chemiluminescent systems. Recently Zhou et al. described that ZnS QDs could enhance chemiluminescence (CL) signals emitted from interaction of NaClO with H2O2 in basic medium [32]. Xiao's group prepared different types of ZnS QDs and have reported their effects on H2O2–NaIO4 CL system [33]. However, the sensitized CL of ZnS QDs on Ce(IV)–SO32− system has not been reported. The oxidation of sulfite in acidic solutions using cerium (IV) or KMnO4 is an important CL reaction, but the CL emission is quite weak. In the present study, we have found that the oxidation of sulfite by Ce(IV) in acidic media and in the presence of ZnS QDs, that act as sensitizers, and atropine, that act as enhancer, produces strong CL signal to allow the development of detection systems. This paper presents a rapid, simple and sensitive method for measuring atropine in pharmaceutical formulation with satisfactory results.
2. Experimental 2.1. Reagents and chemicals All the reagents or solvents were of analytical grade and used without further purification. Ultrapure water (deionized and
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doubly distilled) was used throughout. ZnSO4 7H2O and Lcysteine hydrochloride anhydrous were from Fluka (Buchs, Switzerland). MnCl2 4H2O, Na2SO3 and H2SO4 were purchased from Merck (Darmstadt, Germany). Na2S 9H2O was from Acros (Geel, Belgium). Atropine (99%) and Ce(SO4)2 4H2O were purchased from Sigma-Aldrich. A stock solution of 1.0 10−2 mol L−1 Na2SO3 was prepared daily in water and diluted as required. The solution of 5.0 10−4 mol L−1 Ce(SO4)2 was prepared daily in 0.01 mol L−1 H2SO4 solution. Stock standard solution of atropine (1 10−3 mol L−1) was prepared by dissolving 0.0289 g atropine in 100 mL distilled water and stored in dark bottles at 4 1C in a refrigerator. Working standard solutions were prepared daily by diluting the stock solutions with distilled water just before use. Atropine injection labeled as containing 0.5 mg mL−1 of atropine (Darou Pakhsh, Tehran, Iran) is available as a drug sample and was used.
2.2. Apparatus X-ray diffraction (XRD) patterns were recorded on a Bruker AXS D8 Advance X-ray diffractometer (Bruker, Germany) with Cu Kα radiation (λ¼1.5418 Å). Size distribution of Mn-doped ZnS QDs was performed on a Hydrosol Nanoparticle size analyzer and a Zeta Potential Analyzer (Malvern, UK) which is based on a dynamic light scattering (DLS) technique. The FT-IR spectra (4000–400 cm−1) in KBr were recorded using an FT-IR spectrometer (Tensor 27- Bruker). UV– vis absorbance spectra of Mn-doped ZnS nanocrystals were obtained from aqueous Mn-doped ZnS QDs solutions using a Cecil CE5501 spectrophotometer (Cambridge, UK). Photoluminescence (PL) measurements were recorded on a Perkin-Elmer LS-3B Luminescence Spectrometer (Waltham, USA) using 10 mm quartz cuvettes. The CL light intensity time curve was obtained on Berthold detection systems, Sirius-tube luminometer (Pforzheim, Germany). All optical measurements were carried out at room temperature.
2.3. Synthesis of aqueous Mn-doped ZnS quantum dots Colloidal water-soluble Mn-doped ZnS QDs were synthesized via arrested precipitation in water as described previously with slight modifications [34]. Briefly, 50 mL of 0.02 M L-cysteine, 5 mL of 0.1 M ZnSO4 and 1.5 mL of 0.01 M MnCl2 were added into a three-necked flask. The mixed solution was adjusted to pH 11 with 1 M NaOH and stirred under dry nitrogen at room temperature for 30 min. Then, 5 mL of 0.1 M Na2S was quickly injected into the solution. The mixture was stirred for another 20 min, and then the solution was aged at 50 1C in air for 2 h to form L-cysteine capped Mn-doped ZnS QDs. Purification of the QDs was carried out by precipitation of the nanoparticles with ethanol in a centrifuge at 5000 rpm for 5 min (the procedure was repeated for three times). The obtained QDs were dried under vacuum and stored as a water soluble brown solid powder. Finally, the purified QDs were redissolved in water for further experiments.
2.4. Procedure 2.4.1. Procedure for calibration Working standard solutions containing atropine in the range of 1 10−9–1 10−6 M were prepared by dilution of a concentrated fresh standard solution of atropine (1 10−3 mol L−1). The CL signal was measured by injecting 50 μL of working standard solution into the mixture of Mn-doped ZnS QDs and Na2SO3 solution (appropriate concentrations in water). The CL emission intensities versus atropine concentration were used for the calibration.
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2.4.2. Procedure for pharmaceutical preparation 50 μL of injection sample (0.5 mg mL−1) was diluted to 100 mL with doubly distilled water to the working range of the determination of atropine, and then used for analysis. 2.4.3. Procedure for CL detection Solution A was made by mixing 100 μL of Mn-doped ZnS QDs (appropriate concentrations in water), 100 μL of Na2SO3 (appropriate concentrations in water) and 50 μL water or 50 μL atropine standard solutions (various concentrations in water). Solution A was delivered to the instrument quartz cuvette via polypropene syringes. The mixture was shaken thoroughly and equilibrated at room temperature for 10 min. Then 100 μL proper concentration of Ce(IV) solution was injected into the quartz cuvette and the chemiluminescence spectrum was recorded.
cores due to the solvation layer around the QDs in aqueous solution [36,37]. In addition, Fourier transform infrared spectroscopy was carried out in order to confirm the bonding of L-cysteine to the nanoparticle surface. Fig. 4 shows the FT-IR spectra of free L-cysteine and L-cysteine-capped Mn-doped ZnS nanoparticles. The IR absorption band around 1550–1600 cm−1 (sυ COO−), 1400 cm−1 (mυ COO−), 3000–3500 cm−1 (mυ OH, COOH), indicate the –COO− group. The peak at 2900–3420 cm−1 (mυ N–H) indicates –NH2 group and 600–800 cm−1 (wυ C–S) indicate the C–S group, while the peak at 2550–2750 cm−1 (wυ S–H) represents –S–H group. Results showed that the stretching band of the S–H thiol group, (2550–2670 cm−1 wυ S–H), is not observed when the nanoparticles are evaluated. The reason for disappearance of S–H group vibration on the surface of ZnS nanoparticles is due to the formation of covalent bonds between thiols and Zn2+ surface atoms.
3. Results and discussion 3.2. Spectral characteristics of Mn-doped ZnS QDs 3.1. Characterization of Mn-doped ZnS QDs The XRD pattern of Mn-doped ZnS QDs exhibited a cubic structure with some peaks for (311), (220), and (111) planes (Fig. 2). No diffraction peaks from manganese impurities were detected. The average size (D) of ZnS nanoparticles can be calculated according to Scherrer's equation [35]: D ¼k(λ/β cos θ), where k is a constant (shape factor, about 0.89), λ is the X-ray wavelength (0.15418 nm), β is the full width at half maximum and θ is the diffraction angle. Based on the full width at half-maximum of (111) reflection, the averaged crystallite sizes of Mn:ZnS QDs were estimated to be 4 nm approximately. The size and size distribution of Mn-doped ZnS QDs were also examined by DLS. Fig. 3 shows the scattering intensity distribution of nanocrystals dispersed in ultrapure water at room temperature. The pH of this solution was 6.9. It was found to be aggregate free. The average hydrodynamic size determined from the DLS data was 11 nm, thus suggesting that the nanoparticles dispersed well in water. The hydrodynamic diameters were larger than those of the
Water-soluble L-cysteine-capped Mn-doped ZnS QDs are optically characterized by UV–vis absorption spectroscopy and fluorometry. The characteristic absorption peak of L-cysteine-capped Mn-doped ZnS QDs occurs in 290 nm as shown in Fig. 5a. For all the doped samples, two different emission bands dominated the fluorescence spectra (Fig. 5b). The first emission band at about 460 nm also existed in the FL spectrum of the undoped ZnS nanocrystals, this emission band should originate from the host ZnS. Indeed it is related to ZnS surface defects but not from Mn2+ ions. Upon Mn2+ doping, a second characteristic emission band centered at around 582 nm, is developed for the well-known 4 T1-6A1 d–d transition of Mn2+ ions on Zn2+ sites, where Mn2+ is tetrahedrally coordinated by S2− [17,18].
Fig. 4. FT-IR spectra of free L-cysteine and L-cysteine-capped Mn-doped ZnS QDs.
Fig. 2. XRD patterns of Mn-doped ZnS QDs aged at 50 1C for 2 h.
Fig. 3. Particle size distribution of Mn-doped ZnS QDs measured by DLS (pH¼ 6.9).
Fig. 5. Absorption (a) and fluorescence (b) spectra of L-cysteine-capped Mn-doped ZnS QDs λex ¼ 290 nm.
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Sooklal et al. [38] found that Mn2+ incorporated into the ZnS lattice led to the Mn2+-based orange emission while ZnS with surface-bound Mn2+ yielded the ultraviolet emission. Thus, it could be concluded that the Mn2+ ions in our samples were indeed incorporated into the host ZnS nanocrystals. Photoluminescence quantum yields (QY) were estimated by comparison of the fluorescence intensity with standard dye solutions with the same optical density at the excitation wavelength and similar fluorescence wavelength [39]. The QY values were calculated by the equation: QYðsampleÞ ¼ ðF sample =F ref ÞðAref =Asample Þðn2sample =n2ref ÞQYðrefÞ
ð1Þ
where F, A and n are the measured fluorescence (area under the emission peak), the absorbance at the excitation wavelength, and the refractive index of the solvent, respectively. PL spectra were spectrally corrected and quantum yields were determined relative to rhodamine 6G in water (QY ¼95%) [40]. The PL quantum yield of 13.0% was calculated for Mn-doped ZnS QDs. 3.3. Sensitized effect of Mn-doped ZnS QDs on Ce(IV)–SO32− CL Chemiluminescence emission of Mn-doped ZnS QDs was studied in Ce(IV)–SO32−–ZnS QDs system. It was reported that the oxidation of sulfite by Ce4+ in acidic medium yields a weak chemiluminescent emission, which can be enhanced in the presence of sensitizers or fluorophore compounds, one of which is QDs that attract special attention due to their high quantum yields [27–30]. Therefore, in this study we investigate the effects of ZnS NCs on the Ce(IV)–SO32− CL system. Fig. 6 shows the dynamic CL intensity–time profiles of the Ce (IV)–SO32− (curve a) and Ce(IV)–SO32−–ZnS NCs (curve b) were acquired in the static-injection mode. It indicated (Fig. 6b) that the CL reaction was very quick and the CL intensity reached a maximum in about a second after the injection. It could be seen from Fig. 6 that the CL intensity of Ce(IV)–SO32−–ZnS NCs system is far stronger than that of Ce(IV)–SO32− system, indicating the great sensitized effect of ZnS NCs on Ce(IV)–SO32− CL reaction. Parameters influencing the CL signals of Ce(IV)–SO32−–ZnS NCs system were then investigated to establish the optimal conditions for the CL reaction. This optimization was carried out in the following experiment. 3.4. Optimization of the reaction conditions A series of experiments were conducted for the optimization of Ce(IV)–SO32−–ZnS QDs CL system. In order to perform optimization analysis, four factors were evaluated, including the H2SO4 concentration in Ce(IV) solution, sulfite concentration, Ce(IV)
Fig. 6. Dynamic CL intensity–time profiles of Ce(IV)–SO32− (a), and Ce(IV)–SO32−– ZnS QDs (b). Conditions: 100 μL 5.0 10−4 M Ce(IV) in 0.01 M H2SO4 was injected into a mixture of 100 μL 2.5 10−3 M SO32− plus 150 μL water (a), and 100 μL 5.0 10−4 M Ce(IV) in 0.01 M H2SO4 was injected into a mixture of 100 μL 2.5 10−3 M SO32− plus 100 μL 50 mg L−1 ZnS QDs solution plus 50 μL water (b).
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concentration and ZnS QDs concentration. The CL emission intensity of QDs (peak height) was considered as the experimental response. In sulfuric medium, cerium(IV) is highly stable and does not require any special precaution to prevent the decomposition, but acidity plays a pronounced influence on the development of the chemical reactions. This influence was evaluated within 0.001– 0.15 mol L−1 H2SO4. As shown in Fig. 7a by increasing the acidity (0.01–0.15 mol L−1 H2SO4) a proportional reduction in the analytical signal was observed. The highest CL emission peak heights were achieved at 0.01 mol L−1 H2SO4. The effect of sulfite concentration on CL emission was investigated over the range of 0.01–10 mmol L−1. As shown in Fig. 7b the analytical signal exhibited a pronounced increase up to a concentration value of 1.0 10−3 mol L−1 and then approached stabilization. Therefore, the optimum concentration of SO32− was selected as 1.0 10−3 mol L−1. The effect of Ce(IV) concentration on the CL intensity was examined from 0.01 to 2.0 mmol L−1 in 0.01 mol L−1 H2SO4 (Fig. 7c). Concentrations lower and higher than 5.0 10−4 mol L−1 Ce led to a reduction in the intensity of the analytical signal so Ce (IV) concentration was then selected as 5.0 10−4 mol L−1 Ce as the optimum concentration. The response of different concentrations of ZnS QDs to the present CL system was investigated under the optimal reaction conditions. As shown in Fig. 7d the analytical signal increased with increasing QD concentrations, but for the highest concentrations the repeatability was impaired. Use of 20 mg L−1 ZnS QD solution led to reproducible signals (r.s.d. 1.5%). The ZnS QDs concentration was then fixed as 20 mg L−1, which assured a compromise between sensitivity, analytical dynamical concentration range and precision.
3.5. Analytical applications 3.5.1. Calibration curves and performance characteristics By adding different amounts of atropine in proposed chemiluminescence system (Ce(IV)–SO32−–ZnS QDs) changes in chemiluminescence intensities (ΔICL) are quantitatively related to the concentration of atropine. Under the optimal experimental conditions described above, the calibration graph (i.e., the relationship between the concentration of atropine and the changes in the intensities) was obtained (Fig. 8, inset) and following results were achieved: the regression equation is ΔICL ¼811553C+40,378 (where C is the concentration of atropine, in mmol L−1) with correlation coefficient (r) of 0.9992, the linear range is 1 10−9– 1 10−6 mol L−1 and the detection limit (S/N ¼3) is 2.54 10−10 mol L−1 atropine (Fig. 8). From Table 1, it can be seen that the proposed method has a lower detection limit and wider linear range, compared with most of other methods.
3.5.2. Sample determination and recovery tests To test the applicability of the proposed method, it was applied to the analysis of atropine in the atropine injection. The samples were diluted appropriately with water before measurement. The results are shown in Table 2. As can be seen, the RSD was 2.6% and the recovery of the real samples was 98.2%, which suggested that there were no significant differences between the compared values, making this new chemiluminescence method applicable to these pharmaceutical formulations. Recovery tests were done to estimate the accuracy of this method. So a known amount of standards was added to injection sample in three different levels. Results are given in Table 3. The recoveries ranged from 98.87% to 101.01%, with RSDs of o 4%. It indicated that the proposed method was reliable.
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Fig. 7. Effects of the experimental conditions on the Ce(IV)–SO32−–ZnS QDs CL system. (a) Effects of H2SO4 concentration in Ce(IV) solution: 2.0 10−4 M Ce(IV), 1.0 10−3 M SO32−, 30 mg L−1 ZnS QDs, (b) effects of sulfite concentration: 2.0 10−4 M Ce(IV) in 0.01 M H2SO4, 30 mg L−1 ZnS QDs, (c) effects of Ce(IV) concentration in 0.01 M H2SO4: 1.0 10−3 M SO32−, 30 mg L−1 ZnS QDs, and (d) effects of ZnS QDs concentration: 5.0 10−4 M Ce(IV) in 0.01 M H2SO4, 1.0 10−3 M SO32−.
that Ce(IV) can react with atropine rapidly and completely. In order to check whether the reaction between Ce(IV) and atropine can produce CL, the dynamic CL intensity–time profiles of Ce(IV)–atropine were obtained. But the CL test showed that there was no CL phenomenon in the reaction of Ce(IV) and atropine [7], so participation of their reaction product in the chemiluminescence light producing pathway was judged to be negligible.
3.7. Possible CL mechanism
Fig. 8. The changes of the CL spectra of Ce(IV)–SO32−–ZnS QDs system after addition of various concentrations of atropine. The solution conditions were: 100 μL 5.0 10−4 M Ce(IV) in 0.01 M H2SO4 was injected into a mixture of 100 μL 2.5 10−3 M SO32− plus 100 μL 50 mg L−1 ZnS QDs solution with different concentrations of atropine: (1) 0.0, (2) 0.001, (3) 0.01, (4) 0.05, (5) 0.1, (6) 0.25, (7) 0.5, (8) 0.75, and (9) 1 μmol L−1. The inset shows linear dependence of relative chemiluminescence intensity ΔICL as a function of atropine concentration (μmol L−1).
3.6. Fluorescence analysis In order to explore the reaction mechanism, the FL spectra of atropine and Ce(IV)–atropine mixture were investigated. From the experiment (Fig. 9a), it can be seen that atropine is a fluorescent substance and its fluorescence emission peak is observed at 289 nm. The change of fluorescence emission spectra after mixing atropine with Ce(IV) in acidic solution was investigated. As seen in Fig. 9b, when the Ce(IV) oxidant solution was injected into atropine solution in a molar ratio 1:1, the maximum emission of atropine at 289 nm declined quickly to the extent of disappearance. It confirmed
The mechanism of CL of Ce(IV)–Na2SO3 system has been extensively examined. The oxidation of sulfite by Ce4+ in acidic medium yields a weak chemiluminescent emission, which has been attributed to the formation of excited sulfur dioxide molecules (SO2n) which radiate during deexcitation [41–44]. This weak chemiluminescent emission can be enhanced by energy transfer to sensitizers or fluorophore compounds. Several compounds can be used, and special attention has been given to QDs and nanoparticles due to their high quantum yields [27–30]. In the literature there are only few studies which exploit the CL of Ce(IV)–SO32−– QDs system. A CL resonance energy transfer between SO2n as donors and QDs as acceptors occurred, and QDs were excited and gave the CL emission when it returned to its ground state. So the excited state of QDs was the emitter in this CL system [28,29]. Recently the possible enhancement mechanism of atropine sulfate on post-chemiluminescence (PCL) of Ce(IV)–SO32− system has been also investigated [7]. The results showed that Ce(IV) can react with atropine sulfate. The reaction between Ce(IV) and atropine sulfate released energy and the sulfur dioxide existing in the solution absorbed the energy and was excited to the excited state [7]. Another approach was proposed by Snyder and Wooten
S.N. Azizi et al. / Journal of Luminescence 144 (2013) 34–40
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Table 1 Comparison of the linear ranges and detection limits for atropine assay in pharmaceutical formulation by the proposed method and other reported methods. Methods
DL (mol L−1)
LDR (mol L−1)
Ref.
Liquid chromatography (LC) Liquid chromatography–mass spectrometry (LC–MS) Electrochemical method Post-chemiluminescence (PCL) Electrochemiluminescence (ECL) Capillary electrophoresis (CE) This work
3.45 10−8 5.77 10−10 1.55 10−9 5.75 10−7 1 10−7 0.5 10−6 2.54 10−10
8.63 10−8–3.45 10−6 3.45 10−9–3.45 10−6 1.37 10−8–9.41 10−8 1.43 10−6–7.19 10−5 1 10−7–1 10−4 0.5 10−6–50 10−6 1 10−9–1 10−6
[4] [5] [6] [7] [8] [9]
Table 2 Determination of atropine in pharmaceutical sample by the proposed method. Formulation
Claimed value (mg/ml)
Found (mg/ Recovery ml) (%)
RSD (n¼ 3, %)
Atropine injection (0.5 mg/ml)
0.5
0.491
2.6
98.2
Table 3 Results of atropine determination and recoveries in pharmaceutical formulation. Sample Added (nmol L−1) Observed (nmol L−1) Recovery (%) RSD (n ¼3, %) 1 2 3 4
0 5 10 20
150 153 158.6 172
– 98.87 99.12 101.01
– 3.2 2.4 1.8
Fig. 9. Fluorescence spectra of atropine and the resultant of Ce(IV)–atropine reaction. (a) 1.0 10−5 M atropine; and (b) the mixture of 5.0 10−4 M Ce(IV) (in 0.01 M sulfuric acid) and 1.0 10−5 M atropine (volume ratio 1:1), λex ¼ 250 nm.
which was a CL reaction of SO2 with atomic oxygen under following reaction: O+O+SO2-O2+SO2+hυ The maximum emission is near 300 nm. Therefore the excited SO2 has enough energy and can transfer it to the quantum dots and led to an intense CL signal [45]. On the basis of literature data [7,27–30] and considering the results of previous section the possible mechanism of the enhancement of Ce(IV)–SO32−–ZnS QD CL system by atropine can be expressed as following: 8 CeðIVÞ þ HSO3 − -HSO3 d þ CeðIIIÞ > > > > < 2HSO3 d -S2 O6 2− þ 2Hþ ð2Þ > S2 O6 2− -SO4 2− þ SO2 n > > > n n : SO þ ZnS QDs-SO þ ðZnS QDsÞ 2 2 8 > < CeðIVÞ þ atropine-product þ energy ðEÞ SO2 þ E-SO2 n > : SO n þ ZnS QDs-SO þ ðZnS QDsÞn 2
ð3Þ
determination of atropine (1.0 10−8 mol L−1). The tolerable molar concentration ratios with respect to 1.0 10−8 mol L−1 atropine were more than 500 for K+, Na+, Cl−, glucose, stearic acid, starch, and lactose, 100 for Ca2+, Zn2+, Ni2+, Mg2+ and Mn2+, 20 for uric acid and 10 for Cu2+, Fe3+ and urea.
4. Conclusion This paper describes for the first time the sensitized effect of ZnS QDs on Ce(IV)–SO32− system. The results showed that Mn-doped ZnS QDs can be profitably used as chemiluminescence sensitizers making possible the chemiluminometric determination of compounds that have the potential or that can interact with the nanodots affecting their photochemical properties and/or reactivity. The proposed method has been applied to the determination of low levels of atropine in pharmaceutical products. Compared to the present method for determination of atropine, the proposed method showed its advantages in simplicity, sensitivity and wide linear range.
2
References (ZnS QDs)n-ZnS QDs+hυ
(4)
3.8. Interference studies In order to evaluate possible interferences in this system, the effects of some inorganic ions and organic compounds, on the chemiluminescence intensity of the Mn-doped ZnS QDs system containing 1.0 10−8 mol L−1 atropine were investigated. The tolerance limit was described as the amount of foreign substances which caused relative error less than 7 5% (RSD) in the
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Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Determination of atropine using Mn-doped ZnS quantum dots as novel luminescent sensitizers Seyed Naser Azizi a, Mohammad Javad Chaichi a,n, Parmis Shakeri a, Ahmadreza Bekhradnia b a b
Analytical Division, Faculty of Chemistry, University of Mazandaran, Babolsar 4741695447, Iran Pharmaceutical Sciences Research Center, Department of Medicinal Chemistry, Mazandaran University of Medical Sciences, Sari, Iran
art ic l e i nf o
a b s t r a c t
Article history: Received 16 March 2013 Received in revised form 8 May 2013 Accepted 30 May 2013 Available online 26 June 2013
A novel chemiluminescence (CL) method using water-soluble Mn-doped ZnS quantum dots (QDs) as sensitizers is proposed for the chemiluminometric determination of atropine in pharmaceutical formulation. Water-soluble Mn-doped ZnS QDs were synthesized by using L-cysteine as stabilizer in aqueous solutions. The nanoparticles were structurally and optically characterized by X-ray powder diffraction (XRD), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), UV–vis absorption spectroscopy and photoluminescence (PL) emission spectroscopy. It was found that ZnS quantum dots acted as enhancers of the weak CL emission produced upon oxidation of sulfite by Ce(IV) in acidic medium. Trace amounts of atropine improved the sensitize effect of ZnS quantum dots yielding a significant chemiluminescence enhancement of the Ce(IV)–SO32−–ZnS QD system. Therefore, a new CL analysis system was developed for the determination of atropine. Under the optimum conditions, there is a good linear relationship between the relative chemiluminescence intensity and the concentration of atropine in the range of 1 10−9–1 10−6 M of atropine with a correlation coefficient (R2) of 0.9992. The limit of detection of this system was found to be 2.54 10−10 M. This method is not only simple, sensitive and low cost, but also reliable for practical applications. & 2013 Elsevier B.V. All rights reserved.
Keywords: ZnS quantum dot Sensitized chemiluminescence Ce(IV)–sulfite Atropine
1. Introduction Atropine (Fig. 1) is an alkaloid, which is mainly obtained from Atropa belladonna and in much smaller quantities from Datura stamonium [1]. It has been commonly used in pharmaceutical preparations for the treatment of gastrointestinal diseases, cardiopathy and parkinsonism [2]. Atropine is also used to dilate the pupils in eye operations. The most specific use of atropine is an antidote to organophosphate cholinesterase inhibitors, found in certain insecticides and chemical warfare nerve gases [3]. A high dosage of atropine also stimulates the central nerve system. Despite these positive effects of atropine against diseases, it is also a highly toxic compound. For instance, abnormal kidney function may lead to a toxic reaction in patients receiving atropine treatment, therefore the dosage level of atropine in pharmaceutical preparation is generally very low and for this reason it demands sensitive and selective detection techniques. Several analytical methods have been developed for the analysis of atropine in pharmaceutical dosage forms and in biological
n
Corresponding author. Tel./fax: +98 112 534 2350. E-mail address: [email protected] (M.J. Chaichi).
0022-2313/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jlumin.2013.05.054
fluids including liquid chromatography (LC) with UV detector [4] or tandem mass spectrometry [5], electrochemical method [6], post-chemiluminescence (PCL) [7], electrochemiluminescence (ECL) [8] and capillary electrophoresis [9]. As the dosage level of atropine in pharmaceutical preparation is generally very low and its quantitative determination is significant not only in clinical application but also pharmaceutical analysis; it is important to develop simple, sensitive and accurate methods for being able to detect atropine in formulations and biological systems. During the past decade, semiconductor nanocrystals (quantum dots, QDs) have attracted great interest due to their unique optical and electronic properties, originating from their large surface-tovolume ratio and confinement effect, and have proved to be promising in a number of application areas including biological markers [10], light-emitting diodes [11] or solar cells [12]. One important subset of QDs is those doped with a small percentage of dopants to alter their electronic, magnetic and optical properties for various desired applications [13,14]. Recent investigations have evaluated the use of metallic dopants with quantum states remote from the valence and conduction band edges of the semiconductor nanoparticles to generate different radiative processes [15,16]. In this related, Mn-doped ZnS QDs have attracted considerable attention lately
S.N. Azizi et al. / Journal of Luminescence 144 (2013) 34–40
Fig. 1. Chemical structure of atropine.
because doping Mn2+ ions may act as recombination centers for the excited electron–hole pairs and result in strong and characteristic luminescence at longer wavelengths. Mn2+ emission depends on many parameters like radial location of the dopant, surface ligand and surface charge. So upon Mn2+ doping, a characteristic emission band centered at around 570 nm with a neutral ligand to 600 nm for a charged one, is developed for the well-known 4 T1–6A1 d–d transition of Mn2+ ions on Zn2+ sites of the QD (where Mn2+ is coordinated by S2−) [17,18]. These doped QDs (d-dots) having longer luminescence excited state lifetime, minimized selfabsorption and high quantum efficiency [19] that can be used as cadmium-free emitters for biolabeling [10], as efficient donors for the Förster resonance energy transfer [20], or as sensors for organic and inorganic compounds [21]. In addition the optical properties of QDs strongly depend on the nature of their surface thus modifications of the surface with functional groups or biomolecules and the interactions that it could establish with specific analytes can result in dramatic changes in these properties [22]. Therefore, fluorescence or chemiluminescence (CL) based chemical sensing involving QDs have been developed for different chemical species such as ascorbic acid [23], urea [24], folic acid [25], acetone [26] and cysteine [27]. In most QDs applications, the detection is based on signal quenching, while a more newly attention has been focused on signal enhancing, mainly related to QD ability to sensitize different chemiluminescent systems. Sensitized chemiluminescence is an expeditious policy to exploit CL reactions with low quantum efficiencies for analytical purposes. The weak created energy is transferred to a sensitizer, usually an organic fluorophore with high quantum yield, which is able to magnify it. Any species that selectively interacts with the fluorophore could quench or enhance the CL emission. There are only a few reports involving the sensitized chemiluminescence of semiconductor nanocrystals [23,27–31]. To our knowledge, up to know, there are only few reports on the sensitize effect of ZnS QDs on distinct chemiluminescent systems. Recently Zhou et al. described that ZnS QDs could enhance chemiluminescence (CL) signals emitted from interaction of NaClO with H2O2 in basic medium [32]. Xiao's group prepared different types of ZnS QDs and have reported their effects on H2O2–NaIO4 CL system [33]. However, the sensitized CL of ZnS QDs on Ce(IV)–SO32− system has not been reported. The oxidation of sulfite in acidic solutions using cerium (IV) or KMnO4 is an important CL reaction, but the CL emission is quite weak. In the present study, we have found that the oxidation of sulfite by Ce(IV) in acidic media and in the presence of ZnS QDs, that act as sensitizers, and atropine, that act as enhancer, produces strong CL signal to allow the development of detection systems. This paper presents a rapid, simple and sensitive method for measuring atropine in pharmaceutical formulation with satisfactory results.
2. Experimental 2.1. Reagents and chemicals All the reagents or solvents were of analytical grade and used without further purification. Ultrapure water (deionized and
35
doubly distilled) was used throughout. ZnSO4 7H2O and Lcysteine hydrochloride anhydrous were from Fluka (Buchs, Switzerland). MnCl2 4H2O, Na2SO3 and H2SO4 were purchased from Merck (Darmstadt, Germany). Na2S 9H2O was from Acros (Geel, Belgium). Atropine (99%) and Ce(SO4)2 4H2O were purchased from Sigma-Aldrich. A stock solution of 1.0 10−2 mol L−1 Na2SO3 was prepared daily in water and diluted as required. The solution of 5.0 10−4 mol L−1 Ce(SO4)2 was prepared daily in 0.01 mol L−1 H2SO4 solution. Stock standard solution of atropine (1 10−3 mol L−1) was prepared by dissolving 0.0289 g atropine in 100 mL distilled water and stored in dark bottles at 4 1C in a refrigerator. Working standard solutions were prepared daily by diluting the stock solutions with distilled water just before use. Atropine injection labeled as containing 0.5 mg mL−1 of atropine (Darou Pakhsh, Tehran, Iran) is available as a drug sample and was used.
2.2. Apparatus X-ray diffraction (XRD) patterns were recorded on a Bruker AXS D8 Advance X-ray diffractometer (Bruker, Germany) with Cu Kα radiation (λ¼1.5418 Å). Size distribution of Mn-doped ZnS QDs was performed on a Hydrosol Nanoparticle size analyzer and a Zeta Potential Analyzer (Malvern, UK) which is based on a dynamic light scattering (DLS) technique. The FT-IR spectra (4000–400 cm−1) in KBr were recorded using an FT-IR spectrometer (Tensor 27- Bruker). UV– vis absorbance spectra of Mn-doped ZnS nanocrystals were obtained from aqueous Mn-doped ZnS QDs solutions using a Cecil CE5501 spectrophotometer (Cambridge, UK). Photoluminescence (PL) measurements were recorded on a Perkin-Elmer LS-3B Luminescence Spectrometer (Waltham, USA) using 10 mm quartz cuvettes. The CL light intensity time curve was obtained on Berthold detection systems, Sirius-tube luminometer (Pforzheim, Germany). All optical measurements were carried out at room temperature.
2.3. Synthesis of aqueous Mn-doped ZnS quantum dots Colloidal water-soluble Mn-doped ZnS QDs were synthesized via arrested precipitation in water as described previously with slight modifications [34]. Briefly, 50 mL of 0.02 M L-cysteine, 5 mL of 0.1 M ZnSO4 and 1.5 mL of 0.01 M MnCl2 were added into a three-necked flask. The mixed solution was adjusted to pH 11 with 1 M NaOH and stirred under dry nitrogen at room temperature for 30 min. Then, 5 mL of 0.1 M Na2S was quickly injected into the solution. The mixture was stirred for another 20 min, and then the solution was aged at 50 1C in air for 2 h to form L-cysteine capped Mn-doped ZnS QDs. Purification of the QDs was carried out by precipitation of the nanoparticles with ethanol in a centrifuge at 5000 rpm for 5 min (the procedure was repeated for three times). The obtained QDs were dried under vacuum and stored as a water soluble brown solid powder. Finally, the purified QDs were redissolved in water for further experiments.
2.4. Procedure 2.4.1. Procedure for calibration Working standard solutions containing atropine in the range of 1 10−9–1 10−6 M were prepared by dilution of a concentrated fresh standard solution of atropine (1 10−3 mol L−1). The CL signal was measured by injecting 50 μL of working standard solution into the mixture of Mn-doped ZnS QDs and Na2SO3 solution (appropriate concentrations in water). The CL emission intensities versus atropine concentration were used for the calibration.
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2.4.2. Procedure for pharmaceutical preparation 50 μL of injection sample (0.5 mg mL−1) was diluted to 100 mL with doubly distilled water to the working range of the determination of atropine, and then used for analysis. 2.4.3. Procedure for CL detection Solution A was made by mixing 100 μL of Mn-doped ZnS QDs (appropriate concentrations in water), 100 μL of Na2SO3 (appropriate concentrations in water) and 50 μL water or 50 μL atropine standard solutions (various concentrations in water). Solution A was delivered to the instrument quartz cuvette via polypropene syringes. The mixture was shaken thoroughly and equilibrated at room temperature for 10 min. Then 100 μL proper concentration of Ce(IV) solution was injected into the quartz cuvette and the chemiluminescence spectrum was recorded.
cores due to the solvation layer around the QDs in aqueous solution [36,37]. In addition, Fourier transform infrared spectroscopy was carried out in order to confirm the bonding of L-cysteine to the nanoparticle surface. Fig. 4 shows the FT-IR spectra of free L-cysteine and L-cysteine-capped Mn-doped ZnS nanoparticles. The IR absorption band around 1550–1600 cm−1 (sυ COO−), 1400 cm−1 (mυ COO−), 3000–3500 cm−1 (mυ OH, COOH), indicate the –COO− group. The peak at 2900–3420 cm−1 (mυ N–H) indicates –NH2 group and 600–800 cm−1 (wυ C–S) indicate the C–S group, while the peak at 2550–2750 cm−1 (wυ S–H) represents –S–H group. Results showed that the stretching band of the S–H thiol group, (2550–2670 cm−1 wυ S–H), is not observed when the nanoparticles are evaluated. The reason for disappearance of S–H group vibration on the surface of ZnS nanoparticles is due to the formation of covalent bonds between thiols and Zn2+ surface atoms.
3. Results and discussion 3.2. Spectral characteristics of Mn-doped ZnS QDs 3.1. Characterization of Mn-doped ZnS QDs The XRD pattern of Mn-doped ZnS QDs exhibited a cubic structure with some peaks for (311), (220), and (111) planes (Fig. 2). No diffraction peaks from manganese impurities were detected. The average size (D) of ZnS nanoparticles can be calculated according to Scherrer's equation [35]: D ¼k(λ/β cos θ), where k is a constant (shape factor, about 0.89), λ is the X-ray wavelength (0.15418 nm), β is the full width at half maximum and θ is the diffraction angle. Based on the full width at half-maximum of (111) reflection, the averaged crystallite sizes of Mn:ZnS QDs were estimated to be 4 nm approximately. The size and size distribution of Mn-doped ZnS QDs were also examined by DLS. Fig. 3 shows the scattering intensity distribution of nanocrystals dispersed in ultrapure water at room temperature. The pH of this solution was 6.9. It was found to be aggregate free. The average hydrodynamic size determined from the DLS data was 11 nm, thus suggesting that the nanoparticles dispersed well in water. The hydrodynamic diameters were larger than those of the
Water-soluble L-cysteine-capped Mn-doped ZnS QDs are optically characterized by UV–vis absorption spectroscopy and fluorometry. The characteristic absorption peak of L-cysteine-capped Mn-doped ZnS QDs occurs in 290 nm as shown in Fig. 5a. For all the doped samples, two different emission bands dominated the fluorescence spectra (Fig. 5b). The first emission band at about 460 nm also existed in the FL spectrum of the undoped ZnS nanocrystals, this emission band should originate from the host ZnS. Indeed it is related to ZnS surface defects but not from Mn2+ ions. Upon Mn2+ doping, a second characteristic emission band centered at around 582 nm, is developed for the well-known 4 T1-6A1 d–d transition of Mn2+ ions on Zn2+ sites, where Mn2+ is tetrahedrally coordinated by S2− [17,18].
Fig. 4. FT-IR spectra of free L-cysteine and L-cysteine-capped Mn-doped ZnS QDs.
Fig. 2. XRD patterns of Mn-doped ZnS QDs aged at 50 1C for 2 h.
Fig. 3. Particle size distribution of Mn-doped ZnS QDs measured by DLS (pH¼ 6.9).
Fig. 5. Absorption (a) and fluorescence (b) spectra of L-cysteine-capped Mn-doped ZnS QDs λex ¼ 290 nm.
S.N. Azizi et al. / Journal of Luminescence 144 (2013) 34–40
Sooklal et al. [38] found that Mn2+ incorporated into the ZnS lattice led to the Mn2+-based orange emission while ZnS with surface-bound Mn2+ yielded the ultraviolet emission. Thus, it could be concluded that the Mn2+ ions in our samples were indeed incorporated into the host ZnS nanocrystals. Photoluminescence quantum yields (QY) were estimated by comparison of the fluorescence intensity with standard dye solutions with the same optical density at the excitation wavelength and similar fluorescence wavelength [39]. The QY values were calculated by the equation: QYðsampleÞ ¼ ðF sample =F ref ÞðAref =Asample Þðn2sample =n2ref ÞQYðrefÞ
ð1Þ
where F, A and n are the measured fluorescence (area under the emission peak), the absorbance at the excitation wavelength, and the refractive index of the solvent, respectively. PL spectra were spectrally corrected and quantum yields were determined relative to rhodamine 6G in water (QY ¼95%) [40]. The PL quantum yield of 13.0% was calculated for Mn-doped ZnS QDs. 3.3. Sensitized effect of Mn-doped ZnS QDs on Ce(IV)–SO32− CL Chemiluminescence emission of Mn-doped ZnS QDs was studied in Ce(IV)–SO32−–ZnS QDs system. It was reported that the oxidation of sulfite by Ce4+ in acidic medium yields a weak chemiluminescent emission, which can be enhanced in the presence of sensitizers or fluorophore compounds, one of which is QDs that attract special attention due to their high quantum yields [27–30]. Therefore, in this study we investigate the effects of ZnS NCs on the Ce(IV)–SO32− CL system. Fig. 6 shows the dynamic CL intensity–time profiles of the Ce (IV)–SO32− (curve a) and Ce(IV)–SO32−–ZnS NCs (curve b) were acquired in the static-injection mode. It indicated (Fig. 6b) that the CL reaction was very quick and the CL intensity reached a maximum in about a second after the injection. It could be seen from Fig. 6 that the CL intensity of Ce(IV)–SO32−–ZnS NCs system is far stronger than that of Ce(IV)–SO32− system, indicating the great sensitized effect of ZnS NCs on Ce(IV)–SO32− CL reaction. Parameters influencing the CL signals of Ce(IV)–SO32−–ZnS NCs system were then investigated to establish the optimal conditions for the CL reaction. This optimization was carried out in the following experiment. 3.4. Optimization of the reaction conditions A series of experiments were conducted for the optimization of Ce(IV)–SO32−–ZnS QDs CL system. In order to perform optimization analysis, four factors were evaluated, including the H2SO4 concentration in Ce(IV) solution, sulfite concentration, Ce(IV)
Fig. 6. Dynamic CL intensity–time profiles of Ce(IV)–SO32− (a), and Ce(IV)–SO32−– ZnS QDs (b). Conditions: 100 μL 5.0 10−4 M Ce(IV) in 0.01 M H2SO4 was injected into a mixture of 100 μL 2.5 10−3 M SO32− plus 150 μL water (a), and 100 μL 5.0 10−4 M Ce(IV) in 0.01 M H2SO4 was injected into a mixture of 100 μL 2.5 10−3 M SO32− plus 100 μL 50 mg L−1 ZnS QDs solution plus 50 μL water (b).
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concentration and ZnS QDs concentration. The CL emission intensity of QDs (peak height) was considered as the experimental response. In sulfuric medium, cerium(IV) is highly stable and does not require any special precaution to prevent the decomposition, but acidity plays a pronounced influence on the development of the chemical reactions. This influence was evaluated within 0.001– 0.15 mol L−1 H2SO4. As shown in Fig. 7a by increasing the acidity (0.01–0.15 mol L−1 H2SO4) a proportional reduction in the analytical signal was observed. The highest CL emission peak heights were achieved at 0.01 mol L−1 H2SO4. The effect of sulfite concentration on CL emission was investigated over the range of 0.01–10 mmol L−1. As shown in Fig. 7b the analytical signal exhibited a pronounced increase up to a concentration value of 1.0 10−3 mol L−1 and then approached stabilization. Therefore, the optimum concentration of SO32− was selected as 1.0 10−3 mol L−1. The effect of Ce(IV) concentration on the CL intensity was examined from 0.01 to 2.0 mmol L−1 in 0.01 mol L−1 H2SO4 (Fig. 7c). Concentrations lower and higher than 5.0 10−4 mol L−1 Ce led to a reduction in the intensity of the analytical signal so Ce (IV) concentration was then selected as 5.0 10−4 mol L−1 Ce as the optimum concentration. The response of different concentrations of ZnS QDs to the present CL system was investigated under the optimal reaction conditions. As shown in Fig. 7d the analytical signal increased with increasing QD concentrations, but for the highest concentrations the repeatability was impaired. Use of 20 mg L−1 ZnS QD solution led to reproducible signals (r.s.d. 1.5%). The ZnS QDs concentration was then fixed as 20 mg L−1, which assured a compromise between sensitivity, analytical dynamical concentration range and precision.
3.5. Analytical applications 3.5.1. Calibration curves and performance characteristics By adding different amounts of atropine in proposed chemiluminescence system (Ce(IV)–SO32−–ZnS QDs) changes in chemiluminescence intensities (ΔICL) are quantitatively related to the concentration of atropine. Under the optimal experimental conditions described above, the calibration graph (i.e., the relationship between the concentration of atropine and the changes in the intensities) was obtained (Fig. 8, inset) and following results were achieved: the regression equation is ΔICL ¼811553C+40,378 (where C is the concentration of atropine, in mmol L−1) with correlation coefficient (r) of 0.9992, the linear range is 1 10−9– 1 10−6 mol L−1 and the detection limit (S/N ¼3) is 2.54 10−10 mol L−1 atropine (Fig. 8). From Table 1, it can be seen that the proposed method has a lower detection limit and wider linear range, compared with most of other methods.
3.5.2. Sample determination and recovery tests To test the applicability of the proposed method, it was applied to the analysis of atropine in the atropine injection. The samples were diluted appropriately with water before measurement. The results are shown in Table 2. As can be seen, the RSD was 2.6% and the recovery of the real samples was 98.2%, which suggested that there were no significant differences between the compared values, making this new chemiluminescence method applicable to these pharmaceutical formulations. Recovery tests were done to estimate the accuracy of this method. So a known amount of standards was added to injection sample in three different levels. Results are given in Table 3. The recoveries ranged from 98.87% to 101.01%, with RSDs of o 4%. It indicated that the proposed method was reliable.
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S.N. Azizi et al. / Journal of Luminescence 144 (2013) 34–40
Fig. 7. Effects of the experimental conditions on the Ce(IV)–SO32−–ZnS QDs CL system. (a) Effects of H2SO4 concentration in Ce(IV) solution: 2.0 10−4 M Ce(IV), 1.0 10−3 M SO32−, 30 mg L−1 ZnS QDs, (b) effects of sulfite concentration: 2.0 10−4 M Ce(IV) in 0.01 M H2SO4, 30 mg L−1 ZnS QDs, (c) effects of Ce(IV) concentration in 0.01 M H2SO4: 1.0 10−3 M SO32−, 30 mg L−1 ZnS QDs, and (d) effects of ZnS QDs concentration: 5.0 10−4 M Ce(IV) in 0.01 M H2SO4, 1.0 10−3 M SO32−.
that Ce(IV) can react with atropine rapidly and completely. In order to check whether the reaction between Ce(IV) and atropine can produce CL, the dynamic CL intensity–time profiles of Ce(IV)–atropine were obtained. But the CL test showed that there was no CL phenomenon in the reaction of Ce(IV) and atropine [7], so participation of their reaction product in the chemiluminescence light producing pathway was judged to be negligible.
3.7. Possible CL mechanism
Fig. 8. The changes of the CL spectra of Ce(IV)–SO32−–ZnS QDs system after addition of various concentrations of atropine. The solution conditions were: 100 μL 5.0 10−4 M Ce(IV) in 0.01 M H2SO4 was injected into a mixture of 100 μL 2.5 10−3 M SO32− plus 100 μL 50 mg L−1 ZnS QDs solution with different concentrations of atropine: (1) 0.0, (2) 0.001, (3) 0.01, (4) 0.05, (5) 0.1, (6) 0.25, (7) 0.5, (8) 0.75, and (9) 1 μmol L−1. The inset shows linear dependence of relative chemiluminescence intensity ΔICL as a function of atropine concentration (μmol L−1).
3.6. Fluorescence analysis In order to explore the reaction mechanism, the FL spectra of atropine and Ce(IV)–atropine mixture were investigated. From the experiment (Fig. 9a), it can be seen that atropine is a fluorescent substance and its fluorescence emission peak is observed at 289 nm. The change of fluorescence emission spectra after mixing atropine with Ce(IV) in acidic solution was investigated. As seen in Fig. 9b, when the Ce(IV) oxidant solution was injected into atropine solution in a molar ratio 1:1, the maximum emission of atropine at 289 nm declined quickly to the extent of disappearance. It confirmed
The mechanism of CL of Ce(IV)–Na2SO3 system has been extensively examined. The oxidation of sulfite by Ce4+ in acidic medium yields a weak chemiluminescent emission, which has been attributed to the formation of excited sulfur dioxide molecules (SO2n) which radiate during deexcitation [41–44]. This weak chemiluminescent emission can be enhanced by energy transfer to sensitizers or fluorophore compounds. Several compounds can be used, and special attention has been given to QDs and nanoparticles due to their high quantum yields [27–30]. In the literature there are only few studies which exploit the CL of Ce(IV)–SO32−– QDs system. A CL resonance energy transfer between SO2n as donors and QDs as acceptors occurred, and QDs were excited and gave the CL emission when it returned to its ground state. So the excited state of QDs was the emitter in this CL system [28,29]. Recently the possible enhancement mechanism of atropine sulfate on post-chemiluminescence (PCL) of Ce(IV)–SO32− system has been also investigated [7]. The results showed that Ce(IV) can react with atropine sulfate. The reaction between Ce(IV) and atropine sulfate released energy and the sulfur dioxide existing in the solution absorbed the energy and was excited to the excited state [7]. Another approach was proposed by Snyder and Wooten
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Table 1 Comparison of the linear ranges and detection limits for atropine assay in pharmaceutical formulation by the proposed method and other reported methods. Methods
DL (mol L−1)
LDR (mol L−1)
Ref.
Liquid chromatography (LC) Liquid chromatography–mass spectrometry (LC–MS) Electrochemical method Post-chemiluminescence (PCL) Electrochemiluminescence (ECL) Capillary electrophoresis (CE) This work
3.45 10−8 5.77 10−10 1.55 10−9 5.75 10−7 1 10−7 0.5 10−6 2.54 10−10
8.63 10−8–3.45 10−6 3.45 10−9–3.45 10−6 1.37 10−8–9.41 10−8 1.43 10−6–7.19 10−5 1 10−7–1 10−4 0.5 10−6–50 10−6 1 10−9–1 10−6
[4] [5] [6] [7] [8] [9]
Table 2 Determination of atropine in pharmaceutical sample by the proposed method. Formulation
Claimed value (mg/ml)
Found (mg/ Recovery ml) (%)
RSD (n¼ 3, %)
Atropine injection (0.5 mg/ml)
0.5
0.491
2.6
98.2
Table 3 Results of atropine determination and recoveries in pharmaceutical formulation. Sample Added (nmol L−1) Observed (nmol L−1) Recovery (%) RSD (n ¼3, %) 1 2 3 4
0 5 10 20
150 153 158.6 172
– 98.87 99.12 101.01
– 3.2 2.4 1.8
Fig. 9. Fluorescence spectra of atropine and the resultant of Ce(IV)–atropine reaction. (a) 1.0 10−5 M atropine; and (b) the mixture of 5.0 10−4 M Ce(IV) (in 0.01 M sulfuric acid) and 1.0 10−5 M atropine (volume ratio 1:1), λex ¼ 250 nm.
which was a CL reaction of SO2 with atomic oxygen under following reaction: O+O+SO2-O2+SO2+hυ The maximum emission is near 300 nm. Therefore the excited SO2 has enough energy and can transfer it to the quantum dots and led to an intense CL signal [45]. On the basis of literature data [7,27–30] and considering the results of previous section the possible mechanism of the enhancement of Ce(IV)–SO32−–ZnS QD CL system by atropine can be expressed as following: 8 CeðIVÞ þ HSO3 − -HSO3 d þ CeðIIIÞ > > > > < 2HSO3 d -S2 O6 2− þ 2Hþ ð2Þ > S2 O6 2− -SO4 2− þ SO2 n > > > n n : SO þ ZnS QDs-SO þ ðZnS QDsÞ 2 2 8 > < CeðIVÞ þ atropine-product þ energy ðEÞ SO2 þ E-SO2 n > : SO n þ ZnS QDs-SO þ ðZnS QDsÞn 2
ð3Þ
determination of atropine (1.0 10−8 mol L−1). The tolerable molar concentration ratios with respect to 1.0 10−8 mol L−1 atropine were more than 500 for K+, Na+, Cl−, glucose, stearic acid, starch, and lactose, 100 for Ca2+, Zn2+, Ni2+, Mg2+ and Mn2+, 20 for uric acid and 10 for Cu2+, Fe3+ and urea.
4. Conclusion This paper describes for the first time the sensitized effect of ZnS QDs on Ce(IV)–SO32− system. The results showed that Mn-doped ZnS QDs can be profitably used as chemiluminescence sensitizers making possible the chemiluminometric determination of compounds that have the potential or that can interact with the nanodots affecting their photochemical properties and/or reactivity. The proposed method has been applied to the determination of low levels of atropine in pharmaceutical products. Compared to the present method for determination of atropine, the proposed method showed its advantages in simplicity, sensitivity and wide linear range.
2
References (ZnS QDs)n-ZnS QDs+hυ
(4)
3.8. Interference studies In order to evaluate possible interferences in this system, the effects of some inorganic ions and organic compounds, on the chemiluminescence intensity of the Mn-doped ZnS QDs system containing 1.0 10−8 mol L−1 atropine were investigated. The tolerance limit was described as the amount of foreign substances which caused relative error less than 7 5% (RSD) in the
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