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Simple and Sensitive Paper-Based Device Coupling Electrochemical Sample Pretreatment and Colorimetric Detection Thalita G. Silva,† William R. de Araujo,† Rodrigo A. A. Muñoz,‡ Eduardo M. Richter,‡ Mário H. P. Santana,§ Wendell K. T. Coltro,∥ and Thiago R. L. C. Paixaõ *,† †

Instituto de Química, Universidade de São Paulo, 05508-000, São Paulo, São Paulo, Brazil Instituto de Química, Universidade Federal de Uberlândia, 38400-902, Uberlândia, Minas Gerais, Brazil § Unidade Técnico-Científica, Superintendência Regional do Departamento de Polícia Federal em MG, 38408-680, Uberlândia, Minas Gerais, Brazil ∥ Instituto de Química, Universidade Federal de Goiás, Campus Samambaia, 74690-900 Goiânia, Goiás, Brazil ‡

S Supporting Information *

ABSTRACT: We report the development of a simple, portable, low-cost, high-throughput visual colorimetric paperbased analytical device for the detection of procaine in seized cocaine samples. The interference of most common cutting agents found in cocaine samples was verified, and a novel electrochemical approach was used for sample pretreatment in order to increase the selectivity. Under the optimized experimental conditions, a linear analytical curve was obtained for procaine concentrations ranging from 5 to 60 μmol L−1, with a detection limit of 0.9 μmol L−1. The accuracy of the proposed method was evaluated using seized cocaine samples and an addition and recovery protocol.

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spot tests can be performed using various types of substrates, including polymers and paper. Paper has the advantages of low cost and ready availability and affordability anywhere in the world.9,10 Additionally, white paper provides strong contrast with colored indicators.9 The combination of colorimetric analysis and paper spot tests is powerful as it reduces the use of laboratory equipment, is rapid and inexpensive with minimal sample consumption, and enables portability so that analysis can be performed in remote locations or in places with poor and limited infrastructure.11 Chemical analysis using paper as platform requires isolated test zones that can be established through design of chemical and/or mechanical barriers to limit mixing, through wicking, of the different analytes. The most common methods are printing, stamping, cutting, embossing, origami, and/or chemical modification of the paper.12−17 Each of these techniques has its advantages and disadvantages with regard to cost, possibility to work with organic solvents, flexibility in design, reliability, and, technology needs.16 The current article describes for the first time the development of a simple and accurate wax paperbased analytical method to detect procaine in seized cocaine samples based on diazotisation of procaine, followed by a coupling reaction with chromotropic acid under alkaline

ocaine is one of the most widely used drugs across the world.1 Several laboratory studies have described the effects of cocaine on the body, principally in the central nervous system (CNS) and its chemical dependence. Street cocaine usually differs considerably from pharmaceutical grade cocaine,1 which is used for this purpose.2 However, adverse reactions and other serious health hazards may occur when a drug contains another pharmacologically active component, i.e., an adulterant. Adulterants are deliberately added to increase the bulk, enhance or mimic a pharmacological effect, or even facilitate drug delivery. The adulterants present unintentionally are as a result of poor manufacturing techniques.3 From the reports gathered, adulterants are predominantly substances readily available including caffeine, procaine, phenacetin, paracetamol, and sugars.4 Pharmaceutical formulations are used as adulterants in drugs of abuse, such as cocaine, to mimic the physical properties and pharmacological effects of the drug or to disguise the dilutions made by traffickers to increase the volume of drugs and, thus, their profits.4,5 This type of adulteration is mainly quantified and qualified using high cost instrumentation, such as near-infrared Raman spectroscopy, infrared absorption spectroscopy,6 and gas chromatography with a flame ionization detector.7,8 Colorimetric spot test analysis is a powerful tool for rapid qualitative analysis and can be applied in situations in which a decision is critical, but sophisticated analytical instrumentation or time-consuming procedures cannot be used.9 Colorimetric © 2016 American Chemical Society

Received: January 7, 2016 Accepted: April 22, 2016 Published: April 22, 2016 5145

DOI: 10.1021/acs.analchem.6b00072 Anal. Chem. 2016, 88, 5145−5151

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Analytical Chemistry

Figure 1. Schematic representation for the fabrication process of the lab-on-paper device.

samples to obtain a final concentration in a solution of 0.05 mmol L−1. In order to obtain quantitative data from the colorimetric tests, we used a common scanner and GIMP2 software (www. gimp.org), which converted all images to grayscale, and to obtain the relative intensities of the spot. Interference Study. In order to investigate the selectivity of the sensor, we evaluated the potential interference of some of the major known adulterants/diluents found in seized cocaine samples. We tested the interference of 0.05 mmol L−1 lidocaine (Lid), benzocaine (Ben), phenacetin (Phe), levamisole (Lev), paracetamol (Par), aminopyrine (Amp), and caffeine (Caf) in the color test analysis of procaine under the optimized experimental conditions. Seized Cocaine Composition Analysis. To characterize the samples of seized cocaine, we qualitatively and quantitatively assessed the presence of adulterants in the samples obtained from the Criminalistics Institute of São Paulo using gas chromatography with a flame ionization detector (GC-FID), according to a procedure reported in the literature,7 in collaboration with the Brazilian National Institute of Criminalistics. Electrochemical Pretreatment. In order to eliminate the chemical interference of benzocaine in the procaine colorimetric test, exhaustive electrolysis of the sample was carried out for oxidizing benzocaine. We used a portable potentiostat (DropSens, Oviedo, Spain) with commercial disposable printed carbon electrodes in order to maintain the portability and easiness of the method. In this system, the best conditions obtained, aiming at the maximum oxidation of benzocaine without significantly compromising the procaine, were as follows: application of 1.2 V for 30 min in a 40 μL droplet containing 0.1 mmol L−1 of the compounds, in 0.1 mol L−1 HCl. The solution was directly applied over the electrodes, and after electrolysis, an aliquot (0.5 μL) of this solution was taken for colorimetric spot test analysis. Finally, we propose the integration of the electrochemical system on the filter paper platform, taking advantage of the microfluidic characteristics of this substrate. The electrochemical pretreatment was conducted in controlled conditions; therefore, the device was inserted into a cylindrical glass tube having dimensions of 2.5 cm radius × 6 cm long (internal volume, ∼25 mL) to protect the device from

conditions. The selectivity of the method is enhanced by including an electrochemical sample pretreatment step.

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EXPERIMENTAL SECTION Chemicals and Solutions. All chemicals were of analytical grade and used without additional purification. Phenacetin, lidocaine, procaine, levamisole, paracetamol, and benzocaine were obtained from Sigma-Aldrich (St. Louis, MO). Aminopyrine was obtained from Alfa Aesar (Johnson Matthey Company). Sodium nitrite, sodium hydroxide, chromotropic acid (CTA), and ethanol were purchased from Merck (Darmstadt, Germany). The seized cocaine samples were obtained from the Criminalistics Institute of São Paulo, SP, Brazil, and were manually crushed and homogenized. All powder reagents were solubilized in aqueous media, except benzocaine, lidocaine, phenacetin, and seized cocaine samples, which were solubilized in a mixture of ethanol/water (1:1 v/v). Fabrication of Patterned Paper. The colorimetric spot tests were designed using CorelDRAW X6 software and fabricated using wax printing technology.18 The wax pattern designs were printed on qualitative filter paper using a Xerox wax printer (Norwalk, CT). The specific pattern consisted of white circles with a diameter of 2 mm on a black background. The printed sheets were then placed in a thermal press (Hobby Line Metalno, Santa Catarina, Brazil) for 3 min at 120 °C. Heating causes fusion of the deposited wax on the filter paper, which then penetrates the paper and defines hydrophobic barriers around the pattern. One side of the device was covered with transparent tape to prevent solution leakage through the device and to add structural integrity.19 Colorimetric Analysis. For the spectrophotometric study, absorbance measurements were carried out using a UV−vis spectrophotometer (model U-3000, Hitachi, Tokyo, Japan) with a quartz cuvette with optical path length of 1 cm. In the colorimetric spot tests, an alkaline solution of the chromophore (CTA) was previously pipetted on each spot and it was allowed the solution dry to add the diazotized procaine standard solution or seized cocaine samples. The optimum experimental conditions were 0.5 μL of 2 mg mL−1 CTA + 0.2 mol L−1 NaOH solution and 0.5 μL of 0.8 mmol L−1 NaNO2 + 0.1 mol L−1 HCl solution containing procaine. Recovery tests were performed by adding solid procaine to the real seized cocaine 5146

DOI: 10.1021/acs.analchem.6b00072 Anal. Chem. 2016, 88, 5145−5151

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Scheme 1. Schematic Representation of the Diazotization of Procaine and Coupling with Chromotropic Acid to Form the Colored Compound

Figure 2. (A) Photograph showing colorimetric test results for different procaine concentrations: 0.01 mmol L−1 (a), 0.05 mmol L−1 (b), 0.1 mmol L−1 (c), and 0.2 mmol L−1 (d). (B) Absorbance spectra of the solutions in part A.

barrier. The unprinted zone of the first circle was removed using a hole punch and the screen-printed electrode was attached with a double sided tape. This region was used as sample zone and to perform the electrochemical pretreatment. The second 8 mm circle was printed with channel of 2 mm width and 2 cm long containing one spot in the middle for derivatization process (diazotization reaction) and one in the end for the colorimetric detection. The manifold and

the external environment and control the evaporation during the electrolysis. Fabrication of Lab-on-Paper Device with Electrochemical Pretreatment. We designed and fabricated the device using the same approach used for the colorimetric analysis previously reported, with modification in the printed pattern. The specific pattern consisted of two white circles with a diameter of 8 mm spaced one each other in 1 cm by a wax 5147

DOI: 10.1021/acs.analchem.6b00072 Anal. Chem. 2016, 88, 5145−5151

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Figure 3. (A) Photograph of the colorimetric response of procaine at different concentrations ranging from 0 to 0.4 mmol L−1 in triplicate. (B) Calibration curve obtained from the relative intensities for different procaine concentrations.

with 0.1 mol L−1 HCl, which was selected as the optimal concentration. The effect of CTA concentration on the color intensity was also studied over the range from 0.1 to 2 mg L−1. The best response was obtained at a concentration of 2 mg L−1, enabling better detectability of procaine (Figure S-3, Supporting Information). Finally, we evaluated the optimal concentration of sodium hydroxide for forming the detected colored compound (Scheme 1, compound II). The NaOH concentration was assessed between 0.025 and 0.4 mol L−1. The relative color intensity increased with increasing NaOH concentration up to 0.2 mol L−1 (Figure S-4, Supporting Information), and hence, this was selected as the optimal value. After all chemical reaction parameters had been optimized, we evaluated the influence of the spot diameter on the color intensity of 0.5 μL of 0.2 mmol L−1 procaine solution was utilized in order to maintain a constant number of moles for all spot sizes (1−6 mm). It was observed that the smallest spot generated the highest signal intensity due to the higher density of colored species per unit area (Figure S-5, Supporting Information). However, low reproducibility was associated with control of the spot size, because of to the wax heating step for pattern fabrication on paper. Therefore, the optimum diameter was chosen as 2 mm. An analytical curve was constructed for procaine concentration over the range from 5 to 400 μmol L−1 under the optimized conditions (Figure 3). Linearity was observed from 5 to 60 μmol L−1, and the straight line was in accordance with the equation (relative intensity) = 5.068 + 942.5 (C/mmol L−1), R2 = 0.99). The detection limit was calculated by multiplying by three times the standard deviation of blank measurements and then dividing the resulting value by the slope (sensitivity) of the analytical curve (3σ/slope). The detection and quantification limits were estimated as 0.9 and 3.0 μmol L−1, respectively. Additionally, we evaluated the robustness of the sensor response for the storage of devices in different drying times after addition of reagents on the paper platform. It was observed a good stability of the procaine colorimetric response throughout the period of 2 days as it can be seen in Figure S-6, available in Supporting Information. This achievement allows the previous fabrication of the device for on-site application. Interference Study. Prior to analysis of the seized cocaine samples, we evaluated the potential interference of some of the major known adulterants. We tested the interference of benzocaine (Ben), phenacetin (Phe), paracetamol (Par),

dimensions of the final device for colorimetric detection are displayed in Figure 1.

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RESULTS AND DISCUSSION Spectrophotometric Evaluation. First, we evaluated the protocol based on the diazotization of procaine, followed by a coupling reaction with a chromophore agent, as reported in the literature.20,21 The mechanism of procaine detection involves two steps, as shown in Scheme 1. First, procaine was added to an acidic medium containing sodium nitrite for the diazotisation reaction between the amino group and nitrous acid (generated in situ from sodium nitrite in a strong acid medium), leading to the formation of compound I. Then, the coupling reaction between diazotized procaine hydrochloride and chromotropic acid in an alkaline medium afforded an intense pink, water-soluble dye (compound II) that exhibited maximum absorption at 505 nm. Figure 2A displays the colorimetric response to different procaine concentrations, and Figure 2B shows the absorbance spectra of these solutions. A shift from pale orange to an intense reddish orange color with increasing procaine concentration (Figure 2A) as well as a good linearity between absorbance and concentration of added procaine (Figure 2B) were observed. Performance of the Colorimetric Sensor on Paper. Following the evaluation of the spectrophotometric response, we conducted experiments on paper taking into consideration portability and accessibility of the method for quick and simple analysis in the field. Therefore, chemical parameters that can affect the reaction rate, such as concentrations of acid and sodium nitrite for the diazotization reaction, chromophore (CTA) concentration, and alkalinity of the medium, were optimized for the colorimetric detection of procaine. First, we investigated the effect of nitrite concentration on the colorimetric response of the paper-based sensor. Five different concentrations of sodium nitrite were studied (Figure S-1, Supporting Information). We found that the nitrite concentration which resulted in the best analytical response, i.e., color intensity, was 0.8 mmol L−1. This concentration was therefore used in subsequent experiments. Another parameter that affects the diazotization reaction is the level of acidity. The HCl concentration was therefore evaluated over the range from 0.025 to 0.4 mol L−1 (Figure S-2, Supporting Information). The optimum response was obtained 5148

DOI: 10.1021/acs.analchem.6b00072 Anal. Chem. 2016, 88, 5145−5151

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Figure 4. (A) Photograph of the colorimetric response for (a) procaine, (b) benzocaine, (c) lidocaine, (d) caffeine, (e) aminopyrine, (f) phenacetin, (g) levamisole, and (h) paracetamol at a concentration of 0.05 mmol L−1 under the optimum analytical parameters. (B) Interference test of each adulterant in the presence of procaine. (C) Graphical plot of relative color intensity for interference due to each individual adulterant and the corresponding procaine mixtures.

Figure 5. (A) Photograph of the colorimetric response of procaine and benzocaine, untreated and after electrochemical sample treatment at a concentration of 0.05 mmol L−1 under the optimum analytical parameters. (B) Graphical plot of relative color intensity for treated and untreated samples.

Figure 6. Schematic procedure for procaine analysis using the lab-on-paper device.

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DOI: 10.1021/acs.analchem.6b00072 Anal. Chem. 2016, 88, 5145−5151

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Table 1. Chemical Composition of Seven Seized Cocaine Samples Identified by GC-FID and Recovery Values Obtained by the Proposed Colorimetric Test compounds found in samples of seized cocaine/% (m/m) samples

phenacetina

A B C D E F G

6.4 1.4 0.7 0.7

caffeinea

lidocainea

22.4 30.6 33.3 59.7

1.1 1.4 6.7 21.2 2.8

paracetamola

aminopyrinea

levamisolea

cocainea

recovery of procaineb/%

60.2 16.8 10.7 24.1 16.9 81.6 91.8

93.3 93.3 89.5 100.0 103.8 88.7 97.8

?c ?c 2.7 6.3

1.1

2.0 0.7

a GC-FID results. bProposed colorimetric methodology. Recovery test for procaine added at a final concentration of 0.05 mmol L−1. cCompound was identified but not quantified.

therefore propose a novel device integrating the entire analytical process, from sample pretreatment to the reactions, step by step, to form the monitored colored compound. Figure 6 shows the proposed design of this lab-on-a-paper. Analysis in this device is accomplished by the application of 40 μL of sample solution onto the region of the electrodes (sample zone). After this process, an electrochemical pretreatment which involves the application of 1.2 V for 30 min was performed and then the device is folded overlapping the two 8 mm circular regions (Step 1, Figure 1). Hence, the solution percolates the channel passing through a prior spot to conduct the diazotization step (reaction with nitrite in acid medium previously pipetted and dried on the paper) and finally reaches the detection spot where occurs the coupling reaction between procaine diazotized with CTA in an alkaline medium (also previously impregnated on the paper). The performance and use of these devices can be seen in the recorded videos added in Supporting Information (Video S-1 and Video S-2). After checking for the interference of major known adulterants in seized cocaine and obtaining good selectivity for procaine, we evaluated the accuracy of the device with the addition and recovery protocol. Table 1 summarizes the results of the recovery tests in real seized cocaine samples with defined chemical compositions obtained by chromatography. All samples were spiked with solid procaine to obtain a final concentration of 0.05 mmol L−1 in solution. As can be seen in Table 1, this method afforded sufficiently high levels of recovery and had good accuracy.

levamisole (Lev), aminopyrine (Amp), and caffeine (Caf) for the proposed method (Figure 4A) and the effect of each compound on the color signal of procaine (Pro) (Figure 4B). All of these compounds are well-known adulterants of illicit drugs such as cocaine.3−5,7,22 All possible interferences were first evaluated at a concentration of 0.05 mmol L−1; except for benzocaine, none of these compounds caused variations in the color intensity of procaine by more than 3.5%. An additional experiment using a 1:100 ratio of procaine/interfering species was performed in order to evaluate if the nonpositive interfering species in the first assay could cause variations in the color intensity at higher concentrations (Figure S-7, Supporting Information) and no significant variation of relative intensity was observed for these compounds, even at 100-fold excess. The chemical interference of benzocaine in this method can be explained by the fact that benzocaine, chemically similar to procaine, contains an aromatic primary amine that enables the formation of a diazonium salt, which subsequently undergoes azo coupling with CTA to form the colored compound. Hence, an electrochemical approach was proposed to preferably oxidize the amino group of benzocaine and thus increase the selectivity of the colorimetric method. We first assessed the voltammetric profiles of Pro and Ben using carbon as working electrode; in order to retain the portability and ease of the method, we used a commercial screen-printed electrode (DropSens). Benzocaine undergoes oxidation at a lower potential than procaine, with a difference of about 50 mV. Hence, we evaluated the optimum conditions (applied potential and time of electrolysis) for procaine analysis without significant benzocaine interference. The best results were obtained under the application of 1.2 V voltage for 30 min to a small aliquot (40 μL) of 0.1 mol L−1 HCl before the colorimetric reaction. In this study, we evaluated possible adulteration (1:1 m/m) at a concentration of 0.05 mmol L−1 for Pro and Ben. Figure 5B shows a comparison of the colorimetric response with and without electrochemical pretreatment. After electrochemical pretreatment, the procaine signal intensity dropped by around 10% due to partial oxidation, whereas that for benzocaine showed a pronounced drop of approximately 96% (Figure 5B). Thus, when applying this methodology, a 10% loss in sensitivity was observed but the selectivity was greatly enhanced. Given the microfluidic properties of paper, it is possible to couple the electrochemical system in this substrate either with the electrodes attached on the paper19,23−25 or directly prepared on paper (screen printed on paper).24,26−28 We

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CONCLUSIONS We have demonstrated a simple, portable, low cost, highthroughput visual colorimetric paper-based analytical device that can detect procaine in seized cocaine samples. The interference of most common adulterants found in seized cocaine samples was verified, and a novel electrochemical approach was used for sample pretreatment in order to increase the selectivity. The device has demonstrated excellent analytical parameters, and for this reason, it is a great promise for field analysis in forensic police intelligence.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b00072. Optimization of reaction parameters, spot size, and robustness test of device (PDF) 5150

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(22) Pagano, B.; Lauri, I.; De Tito, S.; Persico, G.; Chini, M. G.; Malmendal, A.; Novellino, E.; Randazzo, A. Forensic Sci. Int. 2013, 231, 120−124. (23) Nie, Z. H.; Nijhuis, C. A.; Gong, J. L.; Chen, X.; Kumachev, A.; Martinez, A. W.; Narovlyansky, M.; Whitesides, G. M. Lab Chip 2010, 10, 477−483. (24) Liana, D. D.; Raguse, B.; Gooding, J. J.; Chow, E. Sensors 2012, 12, 11505−11526. (25) Santhiago, M.; Henry, C. S.; Kubota, L. T. Electrochim. Acta 2014, 130, 771−777. (26) de Araujo, W. R.; Paixao, T. R. L. C. Analyst 2014, 139, 2742− 2747. (27) Kit-Anan, W.; Olarnwanich, A.; Sriprachuabwong, C.; Karuwan, C.; Tuantranont, A.; Wisitsoraat, A.; Srituravanich, W.; Pimpin, A. J. Electroanal. Chem. 2012, 685, 72−78. (28) Dungchai, W.; Chailapakul, O.; Henry, C. S. Anal. Chem. 2009, 81, 5821−5826.

Movie including the lab-on-paper detection with and without the electrochemical pretreatment for a 0.1 mmol L−1 procaine solution (AVI) Movie including the lab-on-paper detection with and without the electrochemical pretreatment for a 0.1 mmol L−1 benzocaine solution (AVI)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +55 11 30919150. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the Brazilian agencies’ CAPES (Grant Number 3359/2014 Pró-Forenses Edital 25/2014), FAPESP (Grant Number 2011/19903-5), and CNPq (Grant Number 444498/2014-1) support of this research.

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REFERENCES

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DOI: 10.1021/acs.analchem.6b00072 Anal. Chem. 2016, 88, 5145−5151