Determination of lapachol in the presence of other naphthoquinones using 3MPA-CdTe quantum dots fluorescent probe

Spectrochimica Acta Part A 100 (2013) 155–160

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Determination of lapachol in the presence of other naphthoquinones using 3MPA-CdTe quantum dots fluorescent probe Ricardo Q. Aucélio a , Ana I. Peréz-Cordovés b , Juliano L. Xavier Lima a , Aurélio Baird B. Ferreira c , Ana M. Esteva Guas b , Andrea R. da Silva a,∗ a

Chemistry Department, Pontifícia Universidade Católica do Rio de Janeiro, 22451-900 Rio de Janeiro, RJ, Brazil Facultad de Química, Universidad de La Habana, 10400 La Habana, Cuba c Chemistry Department, Institute of Exact Sciences, Universidade Federal Rural do Rio de Janeiro, 23851-970 Seropédica, RJ, Brazil b

a r t i c l e

i n f o

Article history: Received 18 October 2011 Received in revised form 20 March 2012 Accepted 5 April 2012 Keywords: Lapachol Fluorescence quenching Nanoparticles CdTe quantum dots

a b s t r a c t 3MPA-CdTe QDs in aqueous dispersion was used as a fluorescent probe for the determination of lapachol, a natural naphthoquinone found in plants of the Bignoniaceae family genus Tabebuia. Working QDs dispersions (4.5 × 10−8 mol L−1 of QDs) was prepared in aqueous media containing Tris–HCl buffer pH 7.4 and methanol (10% in volume). The excitation was made at 380 nm with signal measurement at 540 nm. To establish a relationship between fluorescence (corrected to inner filter effect) and concentration of lapachol, a Stern–Volmer model was used. The linear range obtained was from 1.0 × 10−5 to 1.0 × 10−4 mol L−1 . The limit of detection (xb − 3 sb ) was 8.0 × 10−6 mol L−1 . The 3MPA-CdTe QDs probe was tested in the determination of lapachol in urine, previously cleansed in an acrylic polymer. The average recovery was satisfactory. © 2012 Elsevier B.V. All rights reserved.

1. Introduction In recent years there has been a great interest in developing new luminophores as analytical probes that have their luminescence characteristics (spectrum, lifetime, anisotropy, etc.) changed by the variation of the conditions in the surrounding environment. Luminescent semiconductor quantum dots (QDs) have attracted considerable attention in fluorescence sensing because of their unique optical properties including very intense luminescence, narrow profile with maximum wavelength adjustable by the control of particle size and higher photostability when compared with traditional organic dyes [1,2]. QDs are nanoparticles often composed of elements from groups II–VI (for instance, CdS, CdSe, CdTe) or III–V (for instance, InAs) of the periodic table. The effect of quantum confinement gives rise to these unique optical and electronic properties of QDs that are not available in either discrete atoms or in bulk solids [3,4]. The QDs core is usually coated with organic substances to prevent oxidation and aggregation of the nanostructure, on the other hand, it is very important to coat the nanoparticles to improve optical properties with sharper and symmetric profiles due to the decreasing of surface defects [5]. The presence of surface ligands may enable some degree of selectivity in sensing chemical species

∗ Corresponding author. Fax: +55 21 3527 1637. E-mail address: [email protected] (A.R. da Silva). 1386-1425/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.04.020

as recently demonstrated in the detection of ciprofloxacin in human serum through functionalized cadmium sulfide nanoparticles [6]. Coating the surface also decreases the toxicity of QDs, in special those containing Cd, As and Pb, allowing them to be applied as biological labels [7–9]. Coating also improves the compatibility of QDs in aqueous systems enabling the formation of a stable and homogeneous dispersion [10]. These water compatible QDs are attractive since they can be used as analytical probes [11] to sense chemical species in aqueous solutions with potential applications in both in vivo and in vitro systems [6,12] CdTe QDs are one of the most used since they can be easily synthesized in aqueous medium under mild conditions with particle size easily adjusted by the control of reaction time [13]. On the other hand, there are many reports on the modification of the surface of CdTe QDs [14–16]. For instance, mercaptopropinonc acid (MPA) capping has improved photostability of CdTe/CdS core/shell nanocrystals and improved luminescence signal by minimizing quenching defects from its surface [17]. The quinones represent a large group of substances present in several families of plants, fungi, lichens and bacteria [18]. Quinones are classified according to the basic aromatic system such as benzoquinone, anthraquinones and naphthoquinones [19]. The plants of South America contains many bioactive species, such as lapachol, (2-hydroxy-3-(3-methyl-2-butenyl)-1,4naphthoquinone) (Fig. 1A), which is a natural naphthoquinone found in plants of the Bignoniaceae family [20]. The research with this naphthoquinone started with its isolation from the bark

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O

O

2. Materials and methods

O OH

A

O

B

O

O

O

O

O

C

O

D

O

SO3H

Fig. 1. Structures of naphthoquinones, lapachol (A), ␤-lapachone (B), ␣-lapachone (C) and ␤-lapachone-3-sulfonic acid (D).

of tree known in Argentina as “lapacho” (Tabebuia avellanedae) [21]. Other bioactive naphthoquinones such as ␤-lapachone (Fig. 1B) and ␣-lapachone (Fig. 1C), which are isomers of lapachol, can also be isolated from natural sources, however in insignificant quantities, therefore, the synthesis is the viable route to obtain these isomers. On the other hand, lapachol is the most abundant naphathoquinone and its extraction is quite simple. It represents about 3–7% of the composition of the plant (wood), depending on the species. In Brazil the species of the genus Tabebuia, popularly known as ipe or pau d’arco are commercialized as extracts of iperoxo (alcoholic, tablets and teas) [20,22]. Naphthoquinones present several biological activities acting as anti-inflammatory [23], bactericidal [24], among others [25,26]. Recently de Souza et al. showed that lapachol, ␤-lapachone and ␣-lapachone, present activity against Fusarium oxysporum, a fungi responsible for damaging crops (beans, potatoes, sugarcane) [27]. Although the literature contains methods for readily converting nonfluorescent quinones into highly fluorescent compounds, very few quantitative methods have been reported for the determination of lapachol. The classic procedure is to reduce lapachol with sodium hydrosulfite to form a fluorescent product, unstable in the presence of oxygen. This fluorescent derivative was used to determine lapachol in serum using benzene as solvent [28]. The reduction also was proposed by Alcanfôr et al. using either sodium dithionite or sodium borohydride in ethanolic solutions. The fluorescent product presented excitation spectrum with several shoulders indicating the formation of several reaction products, which can be a problem for the precision of the method since small variations in the reduction conditions may generate different proportion of products. The method was applied in the analysis of pharmaceutical formulations with limit of detection (LOD) in the ␮g L−1 range [29]. Steinert et al. employed HPLC with absorption detection in the UV range to determine naphthoquinones in plant extracts however the authors failed to detect lapachol in the analyzed plant extracts [30]. More recently, Fonseca et al. validated a HPLC method to quantify lapachol in the presence of ␤-lapachone using isocratic elution and absorption detection at 278 nm. The method was applied in simple lapachol preparations with LOD of 0.3 ␮g L−1 (6.4 ng of lapachol in 20 ␮L injection) [31]. In this work, 3MPA-CdTe QDs was used to develop a selective method for the determination of lapachol. The method was based on the quenching of the QDs fluorescence in the presence of the analyte and the selectivity toward ␤-lapachone was evaluated. The method was applied in the analysis of urine samples.

2.1. Apparatus The structures of the synthesized naphthoquinones were confirmed using nuclear magnetic resonance (Bruker AC200 1 H NMR and an Advance 400 1 H 13 C NMR, MA, USA), Fourier transform infrared spectrometer (Perkin-Elmer model 1605, CT, USA) and gas chromatography (Saturn 2000 Gas Chromatographer with mass spectrometry detector, Varian, CA, USA). The absorption spectra were obtained on Perkin Elmer Lambda 19, UV/Vis/NIR double beam spectrometer using 1 cm quartz cuvettes. All fluorescence measurements were made with Perkin Elmer model LS 55 Luminescence spectrometer with solutions placed in 1 cm optical pathlength quartz cuvettes. Fluorescence spectra were acquired using the FL Winlab® software and measurements were performed with 10 nm excitation and emission spectral bandpass and 1500 nm/min scan rate. Excitation was made at 380 nm with signal measurement at 540 nm. Fluorescence lifetime measurements were conducted using a Model HJY 5000M time-resolved fluorescence spectrometer IBH 5000F (Horiba Jobin Yvon, NJ, USA) with excitation using a nanoLED source at 372 nm. Lifetimes were obtained after mathematical deconvolution of the fluorescence decay from the source pulse profile. 2.2. Reagents 3-Mercaptopropionic acid (3MPA), CdCl2 ·2.5H2 O (98%), tellurium powder (200 mesh 99%), sodium borohydride (98%), methacrylic acid (MAA), ethyleneglycoldimethacrylate (EGDMA), 2,2 -azobisisobutyronitrile (AIBN) solution (0.2 mol L−1 in toluene), tris(hydroxymethyl)aminomethane and ammonium sulfate were purchased from Sigma–Aldrich (St. Louis, USA). All solvents were of analytical grade and purchased from Tedia (Rio de Janeiro, Brazil). Ultrapure water was purified in a Milli-Q system from Millipore (Simplicity model 185, MA, USA) and used to prepare all aqueous solutions. Lapachol was extracted according to the procedure described in the literature [21]. ␤-Lapachone, ␣-lapachone and ␤-lapachone-3-sulfonic acid (Fig. 1D) were synthesized following procedures described in the literature [32,33]. All naphthoquinones were purified and characterized by spectroscopic techniques, and the results agree with literature data [34–36]. Nitrogen gas, commercial brand, was obtained from Linde-gases (Rio de Janeiro, Brazil). 2.3. Synthesis of CdTe QDs NaHTe solution and CdTe QDs and were prepared as described in literature [17] but with a slight modification in the reaction time, in order to obtain QDs with fluorescence emission maximum at 540 nm. Briefly, 0.2 mmol of CdCl2 solution and 0.4 mmol of MPA solution were mixed followed by the pH adjustment (pH 10.0) made by small additions of 1.0 mol L−1 NaOH solution under stirring. The solution was placed in a three-necked flask and then purged with N2 . Under stirring, 2.0 mL of freshly prepared NaHTe solution was added, through a syringe, into the CdCl2 solution at room temperature. The ratio of Cd:MPA:Te in the reaction solution was 1:2:0.2. Then the reaction mixture was heated to reflux temperature 100 ◦ C under inert atmosphere for 180 min. The concentration of CdTe QDs was estimated using the Lambert Beer’s law, that relates the absorbance at the first excitonic peak with the molar extinction coefficient of QDs (ε = 10.043 (D)2.12 ) [37]. The concentration of stock aqueous dispersion of CdTe QDs (considering if it was a real solution) was 1.5 × 10−5 mol L−1 . The size of the nanocrystals was determined from Eq. (1), where D (nm) is the particle size and  (nm) is the wavelength of

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Fig. 2. Absorption spectrum of 3MPA-CdTe QDs (5 × 10−6 mol L−1 ) (A) and (B) fluorescence spectrum of 3MPA-CdTe (4.5 × 10−8 mol L−1 ).

first excitonic absorption peak [37]. Absorption spectrum, shown in Fig. 2A, presents the first excitonic absorption peak at 522 nm, which indicates a CdTe QDs diameter of about 2.8 nm. D = (9.8127 × 10−7 )3 − (1.7147 × 10−3 )2 + (1.0064) − 194.84 (1) The fluorescence profile of the CdTe QDs (excitation at 380 nm) is shown in Fig. 2B, this indicated a symmetric and sharp (58 nm FWHM) emission band with maximum at 540 nm. The quantum yield of 24% was estimated for the 3MPA-CdTe at room temperature. This value was achieved by the comparative approach with a fluorophor standard (fluorescence emission of rhodamine B in ethanol) [38]. 2.4. Synthesis of acrylic polymer The acrylic polymer was synthesized according to procedure reported in literature [39] Briefly, 6.6 mL of dichloromethane, 4.64 mmol of MAA, 23.20 mmol of EGDMA and 0.51 mmol of AIBN (in toluene) were added in a 20 mL glass tube. This solution was cooled on an ice bath and degassed with nitrogen for 10 min before being sealed, under N2 , in a glass ampoule. The polymerization was allowed to proceed at 58 ◦ C for 24 h in a water bath. After this period, the glass ampoule was broken and the monolith obtained was ground mechanically and wet sieved using acetone to obtain regularly sized particles with diameters between 53 and 75 ␮m. 2.5. Preparation of standard solutions and samples The working 3MPA-CdTe aqueous dispersions were prepared in 10.0 mL volumetric flasks by adding 1.0 mL of Tris–HCl 0.02 mol L−1 (pH 7.4), 30 ␮L of the stock aqueous dispersion of 3MPA-CdTe QDs, then, appropriate volumes of the stock naphthoquinone solutions (1.0 × 10−2 mol L−1 in methanol) were added to construct the analyte curves. A complementary volume of methanol was added to achieve 1.0 mL of this solvent in the system. Finally, Milli-Q was added to the volumetric flasks to complete the 10.0 mL volume. The solutions remained at rest for 30 min before measurement. For sample analysis, a specific volume of sample was added to the working QDs dispersion mixed and measured after 30 min. The cleaning of the lapachol fortified urine was performed by the addition of ammonium sulfate solution followed by centrifugation (at 300 rpm). The supernatant was collected and submitted

to solid-phase extraction (SPE) using a laboratory-made cartridge, which consisted on 200 mg of the acrylic polymer packed in a 1mL plastic hypodermic syringe coupled to a PTFE membrane filter (0.45 pore size). Sample matrix components were eluted with 4 mL of ultrapure water and then, 4 mL of methanol was used for eluting the analyte. The organic solvent of the eluted solution was evaporated under nitrogen flow and the sample was re-dissolved in the 3MPA-CdTe working dispersion. All measurements were made in triplicate.

3. Results and discussion 3.1. Experimental conditions for fluorescence quenching measurements 3-Mercaptopropionic acid (3MPA) is a popular coating material for the preparation of aqueous QDs enabling very stable aqueous dispersions and excellent photostability [2]. Experimental conditions were adjusted in order to obtain a stable fluorescence from the 3MPA-CdTe QDs working aqueous dispersion. These choices were made based on available information in the literature and by performing optimization experiments. First, the volume of the 3MPA-CdTe stock solution to be placed in the working solution was set to 30 ␮L (4.5 × 10−8 mol L−1 estimated final concentration of 3MPA-CdTe QDs) in order to enable intense fluorescence just under the saturation of the instrument detector. In aqueous dispersions with pH values lower than 7 (adjusted to 5.0 and 6.0 by adding HCl or NaOH), CdTe QDs photoluminescence decreases significantly (Fig. 3 lines e and f) and when lapachol (6.0 × 10−5 mol L−1 ) was added in this aqueous dispersions the fluorescence was completely eliminated (line g because of the decreasing of the pH (non-buffered systems). On the other hand, in systems buffered with Tris–HCl with pH adjusted to values higher than 8.0 (up to pH 9), it was observed a significant increase in signal intensity (lines a and b), but the fluctuation of signal over time did not enable to make reliable measurements. However, in dispersions at pH 7.4 (buffered systems about the physiological pH) fluorescence was intense and stable (line c) with relative standard deviation (RSD) under 5%. The addition of lapachol (6.0 × 10−5 mol L−1 ) decreased the fluorescence due to the interaction of the analyte with the fluorescent probe (line d). In this case, after the signal decrease, the fluorescence was stable and the pH value of the system maintained constant. Two amounts of the Tris–HCl buffer (1.0 mL of either 0.02 or 0.05 mol L−1 in a final volume of 10.0 mL) were evaluated and no differences in

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Fig. 4. Absorption spectrum of 3MPA-CdTe QDs (4.5 × 10−8 mol L−1 , line a) and 3MPA-CdTe QDs in the presence of lapachol (1 × 10–5 to 1 × 10−4 mol L−1 , lines b–h).

Fig. 3. Fluorescence stability of 3MPA-CdTe QDs (4.5 × 10−8 mol L−1 ) in function of time is systems at different pH values: (a) pH 9.0, (b) pH 8.1, (c) pH 7.4, (d) pH 7.4 with lapachol, (e) pH 6.0, (f) pH 5.0, and (g) pH 6.0 with lapachol.

signal magnitude and stability were observed, therefore, 1.0 mL of the 0.02 mol L−1 buffer was used. Finally, in order to guarantee total dissolution of the naphthoquinones when they are introduced in the working dispersions (in special ␤-lapachona and ␣-lapachone which are insoluble in water), a fixed final volume of methanol (or 10%, in volume) was included in the system. Therefore, the influence of methanol in the aqueous dispersion was monitored in systems containing 1.0–3.0 mL of methanol (10–30%, in volume). It was observed an increase of fluorescence directly proportional to the increase of volume of methanol. The addition of 1.0 mL promoted an increase of 23% in the intensity and the stability of fluorescence was obtained after 25 min of the final mixing of components. The addition of 2.0 mL and 3.0 mL favored an increase of 31–55%, respectively, however it was required over 100 min to stabilize the system, thus, a total volume of 1.0 mL of methanol (also considering the volume of methanol added in the aliquots of analytes) was chosen to be included in the working dispersions.

from inner filter effect (see Eq. (2)) since A540 varied from 0.014 to 0.162 and A380 varied from 0.017 to 0.071. A Stern–Volmer model [41] could be readily used to establish a relationship between measured fluorescence (F) and concentration of lapachol [Lapachol] as seen in Eq. (3). Fluorescence quenching in the presence of increasing quantities of lapachol (from 1.0 × 10−5 to 1.0 × 10−4 mol L−1 ) can be seen in Fig. 5. Fcorr = F × 10[(A380 +A540 )/2]

(2)

F0 = 1 + Ksv [Lapachol] Fcorr

(3)

The Stern–Volmer constant (Ksv ) calculated through this model was 3.97 × 103 . Fluorescence decay measurements indicated that the lifetime of the fluorescence from the probe in the presence of lapachol (  ) is 2.98 ns, which is the same value measured from the probe in absence of lapachol ( = 2.97 ns). These results in terms of lifetime together with the fact that the quenching follows the Stern–Volmer model, indicates that a static quenching takes place between lapachol and the 3MPA-CdTe QDs. In order to establish a comparison, fluorescence quenching was also studied for ␤-lapachone, one of the isomers of lapachol. The fluorescence decay curve (using corrected values for the fluorescence in the presence of ␤-lapachone, produced a Ksv value of 2.18 × 103 L mol−1 , indicating that lapachol has about two times the quenching effect of a similar amount of ␤-lapachone upon the nanoparticle probe. One of the probable explanations for this behavior is that although lapachol and ␤-lapachone are isomers,

3.2. Fluorescence of CdTe QDs in the presence of naphthoquinones Lapachol is a weak acid with a pKa value of about 6.2 [40], which mean that its hydroxyl group is completely non-protonated when dissolved (at a 10−4 mol L−1 concentration level) in the working 3MPA-CdTe dispersion at pH 7.4. Lapachol also presents a wide absorption profile in the UV–vis range (Fig. 4) characteristic of the naphthoquinones and although the absorbance values (taking into consideration a concentration of 1.0 × 10−4 mol L−1 ) at 540 nm (A540 ) is below 0.17 while at 380 nm (A380 ) is below 0.071, the inner filter effect [41], at the excitation and at the emission wavelengths chosen to measure fluorescence from the nanoparticle, may affect the response of the probe. A sequence of absorption spectra of the nanoparticle in the presence of increasing concentrations of lapachol (Fig. 4) shows a clear contribution of lapachol in the absorption of light at 380 nm. Therefore, such contribution must be taken into consideration when establishing a fluorescence quenching model for the nanoparticle probe. The measured probe fluorescence in presence of lapachol were corrected to eliminate any influence

Fig. 5. Fluorescence quenching of 3MPA-CdTe QDs ((a) 0, (b) 1.0, (c) 3.0, (d) 6.0, (e) 7.0, (f) 8.0, (g) 9.0 × 10−5 mol L−1 , (h) 1.0 × 10−4 mol L−1 of lapachol). The arrow indicates that the intensity decreases with increase in concentration of lapachol.

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Table 1 Evaluation of the quenching effect of lapachol (1.0 × 10−5 mol L−1 ) in the presence of naphthoquinones. Molar proportion lapachol:lapachone

Flapachol /F(lapachol+naphthoquinone)

1:0.5 1:0.6 1:0.7 1:0.8 1:0.9 1:1

␣-Lapachone

␤-Lapachone

␤-Lapachone-3-sulfonic acid

0.99 1.00 1.03 1.03 1.04 1.05

1.00 1.00 1.01 1.02 1.03 1.04

1.00 0.99 1.01 1.02 1.04 1.06

the spatial distribution of atoms in their structures differ in great extent. Lapachol present a phenol group in its structure (nonprotonated in the established experimental conditions) that allow this naphthoquinone to easily interact with the nanoparticle, forming a complex and producing a more effective quenching. In the case of ␤-lapachone, this complexation with nanoparticle would be unlikely. On the other hand, naphthoquinones are good acceptors of electrons that may withdraw electrons from the QDs when these are excited by the light source. This withdraw of the electrons would decrease fluorescence from QDs. 3.3. Analytical figures of merit for the determination of lapachol The linear response was observed up to 1.0 × 10−4 mol L−1 (24 ␮g mL−1 ) of lapachol (R2 = 0.998 and r = 0.999) with an equation model of log (F0 /F) = 3.97 × 103 [Lapachol] − 0.96. The limit of detection (LOD) was 8.0 × 10−6 mol L−1 (1.9 ␮g mL−1 ) which means that using 70 ␮L of sample, the absolute limit of detection is 130 ng of lapachol. The LOD was calculated as the concentration of lapachol able to reduce the average fluorescence of the dispersion of QDs (xb ), also called blank, to a value equals to xb − 3 sb , where sb is the standard deviation of 10 blank measurements. The precision of the lapachol measurement was calculated as the variation of the F0 /F value taking into consideration ten independent solutions (in two different lapachol concentrations). In order to do this, the following equation was used: s(F0 /F) = 2

2

F0 /F × [(sF /F) + sF0 /F0 ) ]1/2 . The s(F0 /F) , in percentage values, was 1.2% and 2.2% at respectively the 3.0 × 10−5 mol L−1 and 6.0 × 10−5 mol L−1 concentration levels. 3.4. Interferences from other naphthoquinones The selectivity of the response toward the presence of other naphthoquinones (␤-lapachone, ␣-lapachone and ␤-lapachone3-sulfonic acid) was evaluated. These naphthoquinones are present in natural sources in much lesser quantities than lapachol, therefore the interference was studied using maximum equimolar proportion between lapachol and each one of these others naphthoquinones. Interference was evaluated by the Flapachol /F(lapachol+naphthoquinone) values, which represents the ratio between the fluorescence of a QDs dispersion containing 1.0 × 10−5 mol L−1 of lapachol (Flapachol ) and the fluorescence of a QDs dispersion containing 1.0 × 10−5 mol L−1 of lapachol and one of the other naphthoquinones in increasing concentrations (F(lapachol+naphthoquinone) ). A difference larger than 5% between these signals (ratio larger of 1.05) characterizes interference caused by effective quenching caused by the other naphthoquinones. In Table 1, the Flapachol /F(lapachol+naphthoquinone) are indicated and a small contribution of the other naphthoquinones in quenching the fluorescence from the QDs dispersion occurs only when they are present in a quantity equivalent to the one of lapachol as indicated by the Flapachol /F(lapachol+naphthoquinone) value of 1.05.

Table 2 Recoveries of lapachol in the analysis of urine samples. Sample 1 2

Concentration (mol L−1 ) −5

6.7 × 10 6.2 × 10−5

Recovery (%) 96.8 ± 4.0% 88.0 ± 8.0%

3.5. Urine analysis Extracts (alcoholic, tablets and teas) containing lapachol have been commercialized for human consumption due to its pharmacological properties. Its main sources contain lapachol in percent quantities while none or negligible amounts of its isomers are present. Lapachol will appear in the urine of consumer where it can be detected. Therefore, the 3MPA-CdTe QDs probe was tested in the determination of lapachol in urine samples. Fluorescence from the urine matrix overlaps the one of 3MPACdTe QDs. In order to enable the analysis, a clean-up procedure using SPE was established. First, to 1.0 mL of lapachol fortified (7.0 × 10−5 mol L−1 ) urine sample, was added 0.2 g of ammonium sulfate to enable the precipitation of part of the protein content. The mixture was stirred and then centrifuged for 15 min. The supernatant was submitted to solid-phase extraction, using the laboratory-made cartridge with acrylic polymer. This cartridge consisted in 200 mg of acrylic polymer packed in a 1.0 mL plastic hypodermic syringe. The cartridges were previously washed before adding the sample with 10 mL of methanol to remove possible impurities followed by 5.0 mL of ultrapure water. After loading the sample into the cartridge, it was washed by passing 4.0 mL of ultrapure water. Then, the analyte was eluted from the cartridge with 4.0 mL of methanol into a volumetric flask. The organic solvent was evaporated under nitrogen flow, and then, the sample was reconstituted in the aqueous 3MPA-CdTe dispersion. It was verified an about 10% loss of analyte during the procedure, probably a residual fraction retained in the polymer. Therefore, the calibration solutions (3.0 × 10−5 , 5.0 × 10−5 , 7.0 × 10−5 and 1.0 × 10−4 mol L−1 ) were also submitted to this SPE procedure. Tests with non-fortified urine indicated that the procedure eliminated the spectral interference imposed to the sample matrix in the 3MPA-CdTe QDs fluorescence. The average recovery (three samples made on triplicate) obtained with the proposed approach was very satisfactory taking into consideration the complexity of the sample (Table 2). 4. Conclusions The determination of lapachol using the fluorescence quenching of the 3MPA-CdTe QDs was demonstrated. The proposed method does not rely on derivatization procedures that enable unstable fluorescent products or mixture of fluorophors. The limit of detection (at the ␮g mL−1 level) was in the same magnitude order of the other analytical methods indicated in the literature. In terms of ultra-trace analysis, the method can detect down to 130 ng of lapachol present in the sample added in the QDs working solution.

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Selectivity in the response toward lapachol does not require the use of HPLC to analyze samples containing lapachol and ␤-lapachone, ␣-lapachone and ␤-lapachone-3-sulfonic acid present at the proportions expected in natural samples. When a SPE procedure was applied for sample clean-up, the interference of matrix was eliminated, enabling the determination successful of lapachol in urine samples. Acknowledgements Aucélio RQ thanks Brazilian agencies CNPq and FAPERJ for scholarships. CAPES is acknowledged for the scholarship granted to da Silva AR. Peréz-Cordovéz AI thanks VRAC-PUC-Rio for scholarship. The authors thank scientific funding from FINEP-MCT, CNPq, FAPERJ and CAPES-PNPD. Aucelio RQ thanks Universidad de la Habana and the Cuban government for the working permission granted to Peréz-Cordovéz AI. Dr. Authors thank Dr. Sônia R. Louro (Physics Department – PUC-Rio) for the lifetime measurements. References [1] W.R. Algar, A.J. Tavares, U.J. Krull, Analytica Chimica Acta 673 (2010) 1–25. [2] C. Carrillo-Carrión, S. Cárdenas, B.M. Simonet, M. Valcárcel, Chemical Communications (2009) 5214–5226. [3] F.O. Silva, L.C.Z. Viol, D.L. Ferreira, J.L.A. Alves, M.A. Schiavon, Quimica Nova 33 (9) (2010) 1933–1939. [4] P. O’Brien, N.L. Pickett, Chemistry of Materials 13 (2001) 3843–3858. [5] H.Y. Acar, R. Kas, E. Yurtsever, C. Ozen, I. Lieberwirth, Journal of Physical Chemistry C 113 (2009) 10005–10012. [6] D. Li, Z.Y. Yan, W.Q. Cheng, Spectrochimica Acta A 71 (2008) 1204–1211. [7] A.M. Derfus, W.C.W. Chan, S.N. Bhatia, Nano Letters 4 (1) (2003) 11–18. ´ H.S. Bazzi, Y. Cuie, R.A.G.R.A. Fortin, F.M. Winnik, D. Maysinger, Journal [8] J. Lovric, of Molecular Medicine 83 (2005) 377–385. [9] I.L. Medintz, H.T. Uyeda, E.R. Goldman, H. Mattoussi, Nature Materials 4 (2005) 435–446. [10] T. Wang, R. Sridhar, A. Korotcov, A.H. Ting, K. Francis, J. Mitchell, P.C. Wang, Colloids and Surfaces A 375 (2011) 147–155. [11] S. Jagadeeswari, M.A. Jhonsi, A. Kathiravan, R. Renganathan, Journal of Luminescence 131 (2011) 597–602. [12] L. Liu, J. Zhang, X. Su, R.P. Mason, Journal of Biomedical Nanotechnology 4 (4) (2008) 524–528. [13] C. Maule, H. Gonc¸alves, C. Mendonc¸a, P. Sampaio, J.C.E. da Silva, P. Jorge, Talanta 80 (2010) 1932–1938. [14] Y.F. Liu, J.S. Yu, Journal of Colloid and Interface Science 333 (2009) 690–698. [15] W. Zhao, Y. Fung, O. Waisum, M.P.L. Cheung, Analytical Sciences 26 (2010) 879–884.

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